Human rotavirus vaccine strains and diagnostics

ABSTRACT

Immunogenic pharmaceutical compositions including a live, attenuated human rotavirus or an inactivated human rotavirus are provided which are useful for inducing an immunological response against the rotavirus in a subject. Methods of inducing an immunological response to a rotavirus in a subject are provided by administering an immunogenic pharmaceutical composition including a live, attenuated human rotavirus or an inactivated human rotavirus described herein.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/461,663, filed Aug. 18, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/320,095, filed Nov. 11, 2011, now U.S. Pat. No.8.822,192, which is a U.S. national stage application ofPCT/US2010/034537, filed May 12, 2010, which claims priority from U.S.Provisional Application No. 61/177,393 filed May 12, 2009, the entirecontent of all of which is incorporated herein by reference.

GOVERNMENT SPONSORSHIP

This invention was made by the Centers for Disease Control andPrevention, an agency of the United States Government.

FIELD OF THE INVENTION

The present invention relates generally to virus vaccine strains as wellas vaccine compositions and methods relating thereto. More specifically,the present invention relates to human rotavirus A vaccine strains,vaccine compositions and methods of use to induce an immunologicalresponse against rotavirus A in a subject.

BACKGROUND OF THE INVENTION

Of the various enteric pathogenic viruses causing severe diarrhea inchildren, rotavirus is the most common causing an average of 611,000deaths per year. Virtually all children are infected by rotavirus by age5. The virus is believed to be highly contagious and has been describedas a “democratic” virus since the infection affects no particularsocioeconomic or geographic group disproportionately. While the majorityof children having access to adequate supportive and palliative medicalcare survive infection with no significant long-term consequences, thenumber of deaths associated with severe diarrhea, vomiting, dehydrationand shock is unacceptable and requires preventative intervention ifpossible.

Rotavirus A is an icosahedral virus in the family Reoviridae with adistinct hub-and-spoke morphology. Particular rotaviruses are classifiedby group, subgroup and serotype according to properties characteristicof the viral capsid proteins. Rotavirus particles contain 3 proteinlayers surrounding the viral genome which consists of 11 segments ofdouble-stranded RNA, each segment encoding a protein. The viral proteinsinclude structural proteins called VPs and nonstructural proteinsdesignated NSPs. A number of the structural proteins are particularlyimportant in eliciting an immune response in a host since these proteinsare present on the outermost surface of the viral particles. Inparticular the proteins VP7 and VP4 both figure prominently in hostimmune response and therefore have also played a central role indevelopment of rotavirus vaccines.

Variants of VP7 and VP4 structural proteins characterize distinctrotavirus A serotypes. In particular, variants of human VP7 areidentified as “G” serotypes including at least serotypes G1, G2, G3, G4,as well as the less common G5, G6, G8, G9, G10, G11, G12, G13 and G14.Variants of the VP4 structural protein are identified as “P” serotypesincluding, P1A, P1B, P2A, P3, P4, P5, P6 and P8. Because intactrotaviruses are characterized by both a VP7 protein and a VP4 protein,individual virus serotypes are named according to the identity of thevariant of these two proteins contained in the particular virus. Forexample, a common rotavirus A contains both G1 and P[8] variants of VP7and VP4, respectively. The G1, P[8] serotype of rotavirus A is one ofthe most common forms of the virus which cause disease worldwide. The G1serotype of rotavirus A is the most common serotype associated withhuman disease worldwide. A number of vaccines have been developed whichuse rotavirus A G1 strains with the goal of developing immunity in ahost against rotavirus A G1 strains as well as rotavirus A strainshaving other serotypes. However, this approach has been limited byimportant differences between the G1 and G2 serotypes. In particular,rotavirus A G2 strains are derived from a different lineage than mostother rotavirus strains. This is demonstrated by nucleic acidhybridization experiments showing that labeled transcripts of the 11gene segments of G2 strains, also known as the DS-1 genogroup, do nothybridize with corresponding nucleic acids from the strains known as theWa genogroup of rotavirus A which includes G1, G3, G4, and G9. The lackof hybridization of these homologous genes indicates that differences inthe encoded proteins, such as the outer capsid proteins VP4 and VP7 andinner capsid protein VP6, are substantial. These genetic differencessupport the observations that individuals infected or immunized using aG1 strain are less likely to show cross protection against G2 strainsthan other strains of the Wa genogroup.

In addition, to the common G1 and G2 rotavirus A strains, a diversity ofhuman rotavirus types is increasingly recognized as contributing toacute severe diarrhea disease worldwide. This diversity underscores theneed for robust vaccines capable of generating immunity against severalstrains. Recently, the United States Food and Drug Administrationsuspended the use of the ROTARIX vaccine citing contaminants in itspreparation. Thus, the number of available vaccines for rotavirus isdeclining at a time when infections remain a serious worldwide problem.Another vaccine, RotaTeq®, appears safe and effective in preventingdiarrhea among children in middle and high income countries and arecurrently licensed and recommended for use in infants throughout theworld. However, the efficacy of this vaccine is reduced in low incomecountries of Africa and Asia.

Thus, there is a continuing need for vaccines against human rotavirus Aof both common and less common types.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a xerographic image of an electron micrograph showing intactisolated rotavirus A virions identified as strain CDC-9, having serotypeP[8], G1;

FIG. 1B is a xerographic image of an electron micrograph showing intactisolated rotavirus A virions identified as strain CDC-66, havingserotype P[4], G2;

FIG. 2 shows a xerographic reproduction of an image of a polyacrylamidegel illustrating RNA profiles of rotavirus A strain CDC-9 isolated froma stool sample (S) and Vero cells (V) and showing typical long (CDC-9)RNA electropherotypes for the rotavirus strain;

FIG. 3 shows a xerographic reproduction of an image of a polyacrylamidegel illustrating RNA profiles of the rotavirus A strain CDC-66 isolatedfrom a stool sample and Vero cells and showing typical short RNAelectropherotypes for this rotavirus strain;

FIG. 4A shows bands of CsCl purified rotavirus particles of rotavirus Astrain CDC-9;

FIG. 4B shows identified structural viral proteins of rotavirus A strainCDC-9 analyzed by SDS-PAGE in comparison to molecular weight markers;

FIG. 5A is a bar graph showing total antibody titers in response tothermally inactivated rotavirus in control and vaccinated mice;

FIG. 5B is a bar graph showing neutralizing antibody titers in responseto thermally inactivated rotavirus in control and vaccinated mice;

FIG. 6 is a bar graph showing total serum antibody responses tothermally inactivated rotavirus formulated with Al(OH)₃ in control andvaccinated mice.

FIG. 7A shows virus shedding in fecal samples of piglets vaccinated withno antigen and with 750 micrograms of aluminum phosphate in 4 animals;

FIG. 7B shows virus shedding in fecal samples from piglets immunizedwith antigen and no adjuvant;

FIG. 7C shows virus shedding in fecal samples of piglets immunized withantigen and adjuvant;

FIG. 7D shows virus shedding measured in fecal samples of pigletsimmunized with buffer only;

FIG. 8A is a bar graph showing rotavirus specific IgG antibody responsein sera of piglets vaccinated with no antigen and with 600 micrograms ofaluminum phosphate (solid bars) or piglets vaccinated with 50 microgramsof antigen and with 600 micrograms of aluminum phosphate (hatched bars);

FIG. 8B is a bar graph showing neutralizing antibody response in sera ofpiglets vaccinated with no antigen and with 600 micrograms of aluminumphosphate or piglets vaccinated with 50 micrograms of antigen and with600 micrograms of aluminum phosphate;

FIG. 9A shows virus shedding in fecal samples of piglets vaccinated withno antigen and with 600 micrograms of aluminum phosphate; and

FIG. 9B shows virus shedding in fecal samples of piglets vaccinated with50 micrograms of antigen and with 600 micrograms of aluminum phosphate.

SUMMARY OF THE INVENTION

A vaccine composition is provided including one or more isolatedrotavirus strains illustratively strain CDC-9 or CDC-66 in combinationwith a pharmaceutically acceptable carrier. An inventive vaccineoptionally includes an adjuvant.

The CDC-9 or CDC-66 strains in an inventive vaccine are optionally liveattenuated rotavirus or inactivated rotavirus.

It is appreciated that an inventive vaccine optionally includes at leasttwo isolated rotavirus strains. The at least two isolated rotavirusstrains each independently have a G group serotype of G1, G2, G3, G4,G5, G6, G7, G8, G9, G10, G11, G12, G13 or G14. Optionally, the at leasttwo isolated rotavirus strains each independently have a P groupserotype of P1A, P1B, P2A, P3, P4, P5, P6, P8, P11, or P12.

An inventive vaccine is optionally administered parenterally or orally.

An isolated rotavirus strain is also provided that is illustrativelyCDC-9 or CDC-66 strain.

An inventive vaccine is provided that includes a pharmaceuticallyacceptable carrier admixed with an isolated rotavirus straincharacterized as having a G1 group serotype and an isolated rotavirusstrain characterized as having a G2 group serotype. The G1 or G2 groupserotype strains optionally each independently have a P group serotypeof P1A, P1B, P2A, P3, P4, P5, P6, P8, P11 or P12. In some embodimentsthe human rotavirus strain characterized as having a G1 group serotypeis CDC-9, or the human rotavirus strain characterized as having a G2group serotype is CDC-66.

A method of inducing an immunological response to a rotavirus in asubject is provided including administering a vaccine compositionincluding a pharmaceutically acceptable carrier admixed with an isolatedhuman rotavirus strain of CDC-9 or CDC-66.

A method of inducing an immunological response to a rotavirus in asubject is provided including administering a vaccine compositionincluding a pharmaceutically acceptable carrier admixed with an isolatedhuman rotavirus strain characterized as having a G1 group serotype andan isolated human rotavirus strain characterized as having a G2 groupserotype.

Also provided is a vaccine including a pharmaceutically acceptablecarrier admixed with a portion of an isolated human rotavirus. Theisolated human rotavirus portion is a peptide or polypeptide includingan amino acid sequence of SEQ ID No. 2; SEQ ID No. 5; SEQ ID No. 8; SEQID No. 11; SEQ ID No. 14; SEQ ID No. 17; SEQ ID No. 20; SEQ ID No. 23;SEQ ID No. 26; SEQ ID No. 29; SEQ ID No. 32; SEQ ID No. 3; SEQ ID No. 6;SEQ ID No. 9; SEQ ID No. 12; SEQ ID No. 15; is SEQ ID No. 18; SEQ ID No.21; SEQ ID No. 24; SEQ ID No. 27; SEQ ID No. 30; SEQ ID No. 33; SEQ IDNo. 71; SEQ ID No. 77; SEQ ID No. 83; SEQ ID No. 89; SEQ ID No. 95; SEQID No. 101; SEQ ID No. 107; SEQ ID No. 113; SEQ ID No. 119; SEQ ID No.125; SEQ No. 131; SEQ ID No. 72; SEQ ID No. 78; SEQ ID No. 84; SEQ IDNo. 90; SEQ ID No. 96; SEQ ID No. 102; SEQ ID No. 108; SEQ ID No. 114;SEQ ID No. 120; SEQ ID No. 126; SEQ ID No. 132; a homolog thereof or afragment thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Novel isolated human rotavirus A strains, vaccines including humanrotavirus A strains, vaccines including a human rotavirus A polypeptideand/or an immunogenic fragment thereof, anti-rotavirus A antibodies andmethods for vaccinating humans against rotavirus A disease are providedaccording to embodiments of the present invention.

Scientific and technical terms used herein are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found defined and used in context in various standardreferences illustratively including J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company,2004; Wild, D., The Immunoassay Handbook, 3rd Ed., Elsevier Science,2005; Gosling, J. P., Immunoassays: A Practical Approach, PracticalApproach Series, Oxford University Press, 2005; Antibody Engineering,Kontermann, R. and Dübel, S. (Eds.), Springer, 2001; Harlow, E. andLane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1988; Ausubel, F. et al., (Eds.), Short Protocols in MolecularBiology, Wiley, 2002; J. D. Pound (Ed.) Immunochemical Protocols,Methods in Molecular Biology, Humana Press; 2nd ed., 1998; B. K. C. Lo(Ed.), Antibody Engineering: Methods and Protocols, Methods in MolecularBiology, Humana Press, 2003; and Kohler, G. and Milstein, C., Nature,256:495-497 (1975); the contents of each of which are incorporatedherein by reference.

Human Rotaviruses

Novel human rotavirus A strains of the present invention areillustratively identified as CDC-9 and CDC-66, fragments thereof orhomologues thereof.

The CDC-9 rotavirus A strain was isolated from a fecal specimen of a5-month boy in Providence, R.I. Human rotavirus strain CDC-9 wascharacterized by RT-PCR using G and P type-specific primers. RT-PCRanalysis indicates that isolated strain CDC-9 is a strain having agenotype P[8], G1. Particular characteristics of CDC-9, itsidentification, isolation, and passages in Vero cells are described byEsona, M D, et al., Human Vaccines, 2010; 6:1-7, the entire contents ofwhich are incorporated herein by reference.

Following isolation from the fecal sample, isolated rotavirus strainCDC9 was adapted to grow in MA104 cells that were prepared and frozenbefore 1980 and have complete documentation. The CDC-9 strain was thenadapted to grow in Vero cells qualified for vaccine production. CDC-9was purified by performing 3 rounds of limiting dilution and afteramplification in Vero cells, was further purified by performing 3 roundsof plaque assays. The isolated strain CDC-9 was passaged 7 and 38 timesin MA104 and Vero cells, respectively (total 45 passages). Theadaptation and all passages are done using standard operation proceduresand certified raw materials and reagents and under the Good LaboratoryPractice Guidelines. Unlike other reference or laboratory strains, theisolated strain CDC-9 has complete passage history and documentation.

The titer of passaged human rotavirus strain CDC-9 is about 10⁷ ffu/ml.

Isolated human rotavirus strain CDC-9 was studied by electron microscopyusing CDC-9 virions isolated from the medium of infected Vero cellcultures. FIG. 1A shows an electron micrograph of isolated CDC-9virions. The micrograph shows the virions to have the morphology typicalof human rotavirus A virions.

Isolated human rotavirus strain CDC-9 was further examined usingpolyacrylamide gel electrophoresis of RNA isolated from the strain. Asshown in FIG. 2, CDC-9 has a typical long RNA electropherotype and theRNA profiles of both the original isolate from the stool and the Veropassaged rotavirus are identical. Also shown in FIG. 2 is a standard forcomparison including an RNA profile of a Wa genogroup human rotavirus.

Isolated human rotavirus strain CDC-9 in stool and Vero cells (passage27) was analyzed by sequence analysis of entire genome.

CDC9 amino acid sequences of proteins encoded by nucleic acids isolatedfrom a stool sample: CDC9 NSP1 aa—stool is SEQ ID No. 2; CDC9 NSP 2aa—stool is SEQ ID No. 5; CDC9 NSP 3 aa—stool is SEQ ID No. 8; CDC9 NSP4 aa—stool is SEQ ID No. 11; CDC9 NSP 5 aa—stool is SEQ ID No. 14; CDC9VP1 aa—stool is SEQ ID No. 17; CDC9 VP 2 aa—stool is SEQ ID No. 20; CDC9VP 3 aa—stool is SEQ ID No. 23; CDC9 VP 4 aa—stool is SEQ ID No. 26;CDC9 VP 6 aa—stool is SEQ ID No. 29; and CDC9 VP 7 aa—stool is SEQ IDNo. 32.

CDC9 nucleotide sequences of nucleic acids isolated from a stool sample:CDC9 NSP1 nt—stool is SEQ ID No. 35; CDC9 NSP 2 nt—stool is SEQ ID No.38; CDC9 NSP 3 nt—stool is SEQ ID No. 41; CDC9 NSP 4 nt—stool is SEQ IDNo. 44; CDC9 NSP 5 nt—stool is SEQ ID No. 47; CDC9 VP1 nt—stool is SEQID No. 50; CDC9 VP 2 nt—stool is SEQ ID No. 53; CDC9 VP 3 nt—stool isSEQ ID No. 56; CDC9 VP 4 nt—stool is SEQ ID No. 59; CDC9 VP 6 nt—stoolis SEQ ID No. 62 and CDC9 VP 7 nt—stool is SEQ ID No. 65.

CDC9 amino acid sequences of proteins encoded by nucleic acids isolatedfrom CDC9 rotavirus at passage 27 isolated from Vero cells: CDC9 NSP1aa—Vero is SEQ ID No. 3; CDC9 NSP 2 aa—Vero is SEQ ID No. 6; CDC9 NSP 3aa—Vero is SEQ ID No. 9; CDC9 NSP 4 aa—Vero is SEQ ID No. 12; CDC9 NSP 5aa—Vero is SEQ ID No. 15; CDC9 VP1 aa—Vero is SEQ ID No. 18; CDC9 VP 2aa—Vero is SEQ ID No. 21; CDC9 VP 3 aa—Vero is SEQ ID No. 24; CDC9 VP 4aa—Vero is SEQ ID No. 27; CDC9 VP 6 aa—Vero is SEQ ID No. 30; and CDC9VP 7 aa—Vero is SEQ ID No. 33.

CDC9 nucleotide sequences of nucleic acids isolated from CDC9 rotavirusat passage 27 isolated from Vero cells: CDC9 NSP1 nt—Vero is SEQ ID No.36; CDC9 NSP 2 nt—Vero is SEQ ID No. 39; CDC9 NSP 3 nt—Vero is SEQ IDNo. 42; CDC9 NSP 4 nt—Vero is SEQ ID No. 45; CDC9 NSP 5 nt—Vero is SEQID No. 48; CDC9 VP1 nt—Vero is SEQ ID No. 51; CDC9 VP 2 nt—Vero is SEQID No. 54; CDC9 VP 3 nt—Vero is SEQ ID No. 57; CDC9 VP 4 nt—Vero is SEQID No. 60; CDC9 VP 6 nt—Vero is SEQ ID No. 63; and CDC9 VP 7 nt—Vero isSEQ ID No. 66.

Nucleotide and amino acid sequences of entire genome from CDC-9rotavirus isolated from stool and infected culture were compared withamino acid and nucleotide sequences of entire genome from reference KUor other G1P8 strains of rotavirus A, as shown herein.

Additionally, as shown in Table 2, CDC-9 genes (except for segment 3)share high sequence identity with the corresponding genes of theprototype P[8], G1 human KU strain.

TABLE 2 Percentages of nucleotide (NT) and deduced amino acid (AA)identity of rotavirus vaccine strain CDC-9 gene segments compared withcognate gene sequences of prototype rotavirus strain KU. CDC-9 Gene % NT% AA VP1 88 96 VP2 95 98 VP3 77 80 VP4 91 94 VP6 91 98 VP7 93 96 NSP1 8381 NSP2 90 94 NSP3 93 95 NSP4 93 94 NSP5 93 94

In addition, changes in nt and aa sequences of entire genome of CDC-9strain from stool to passage 27 in Vero cells have been documented, asshown in Table 3.

TABLE 3 Changes of nt and aa in genes of CDC-9 from stool to passage 27in Vero cells Gene # of nt # of aa segment changes nt position changesaa position NSP1 1 396 A→G 1 122 Q→R NSP2 0 0 NSP3 0 0 NSP4 0 0 0 NSP5 1155C→T 1 45 A→I VP1 0 0 VP2 0 0 VP3 (DS-1) 0 0 VP4 6 161G→A, 5 51G→D,1001C→T, 331S→F, 1101G→A, 364M→I, 1162G→C, 385D→H, 1171A→C, 388I→L2025T→C VP6 1 325C→T 1 101A→V VP7 1 678G→A 0 Total: 10 8

Isolated rotavirus CDC-9 in Vero cells is a reassortant that has all(except segment 3) genes from a KU-like strain. CDC-9 has a segment 3derived from a DS-1 like strain as CDC-9 VP3 shares a high identity withthe cognate gene of DS-1 strain. This reassortment might have occurredduring natural infection or when G1P8 and G2P4 rotaviruses in the fecalspecimen were adapted and passaged in cell culture. Rotavirus VP3 hasbeen described to possess guanylyltransferase and may be involved inviral replication and morphogenesis.

The CDC-66 rotavirus A strain was isolated from a fecal specimen of an11-month girl in Providence, R.I. Human rotavirus strain CDC-66 wascharacterized by RT-PCR using G and P type-specific primers. RT-PCRanalysis indicates that isolated strain CDC-66 is a strain having aserotype P[4], G2.

Following isolation from the fecal sample, isolated rotavirus strainCDC-66 was adapted to grow in MA104 cells that were prepared and frozenbefore 1980 and have complete passage history and documentation. TheCDC-66 strain was then adapted to grow in Vero cells qualified forvaccine production. CDC-66 was purified by performing 3 rounds oflimiting dilution and after amplification in Vero cells, was furtherpurified by performing 3 rounds of plaque assays. The isolated strainCDC-66 was passaged 5 and 40 times in MA104 and Vero cells, respectively(total 45 passages). The adaptation and all passages are done usingstandard operation procedures and certified raw materials and reagentsand under the Good Laboratory Practice Guidelines. Unlike otherreference and laboratory strains, the isolated strain CDC-66 hascomplete passage history and documentation.

The titer of passaged human rotavirus strain CDC-66 is about 10⁷ pfu/ml.

Isolated human rotavirus strain CDC-66 was studied by electronmicroscopy using CDC-66 virions isolated from the medium of infectedVero cell cultures. FIG. 1B shows an electron micrograph of isolatedCDC-66 virions. The micrograph shows the virions to have the morphologytypical of human rotavirus virions.

Isolated human rotavirus strain CDC-66 was further examined usingpolyacrylamide gel electrophoresis of RNA isolated from the strain. Asshown in FIG. 3, CDC-66 has a typical short RNA electropherotype and theRNA profiles of both the original isolate from the stool and the Veropassaged rotavirus are identical. Also shown in FIG. 3 are standards forcomparison including a DNA molecular weight marker III (Roche) in thefar left lane and an RNA profile of rhesus rotavirus, RRV.

Isolated human rotavirus strain CDC-66 in stool and Vero cells (passage27) was analyzed by sequence analysis of entire genome.

CDC66 amino acid sequences of proteins encoded by nucleic acids isolatedfrom a stool sample: CDC66 NSP1 aa—stool is SEQ ID No. 71; CDC66 NSP 2aa—stool is SEQ ID No. 77; CDC66 NSP 3 aa—stool is SEQ ID No. 83; CDC66NSP 4 aa—stool is SEQ ID No. 89; CDC66 NSP 5 aa—stool is SEQ ID No. 95;CDC66 VP1 aa—stool is SEQ ID No. 101; CDC66 VP 2 aa—stool is SEQ ID No.107; CDC66 VP 3 aa—stool is SEQ ID No. 113; CDC66 VP 4 aa—stool is SEQID No. 119; CDC66 VP 6 aa—stool is SEQ ID No. 125; and CDC66 VP 7aa—stool is SEQ ID No. 131.

CDC-66 nucleotide sequences of proteins encoded by nucleic acidsisolated from a stool sample: CDC66 NSP1 nt—stool is SEQ ID No. 68;CDC66 NSP 2 nt—stool is SEQ ID No. 74; CDC66 NSP 3 nt—stool is SEQ IDNo. 80; CDC66 NSP 4 nt—stool is SEQ ID No. 86; CDC66 NSP 5 nt—stool isSEQ ID No. 92; CDC66 VP1 nt—stool is SEQ ID No. 98; CDC66 VP 2 nt—stoolis SEQ ID No. 104; CDC66 VP 3 nt—stool is SEQ ID No. 110; CDC66 VP 4nt—stool is SEQ ID No. 116; CDC66 VP 6 nt—stool is SEQ ID No. 122; andCDC66 VP 7 nt—stool is SEQ ID No. 128.

CDC-66 amino acid sequences of proteins encoded by nucleic acidsisolated at passage 27 isolated from Vero cells: CDC66 NSP1 aa—Vero isSEQ ID No. 72; CDC66 NSP 2 aa—Vero is SEQ ID No. 78; CDC66 NSP 3 aa—Verois SEQ ID No. 84; CDC66 NSP 4 aa—Vero is SEQ ID No. 90; CDC66 NSP 5aa—Vero is SEQ ID No. 96; CDC66 VP1 aa—Vero is SEQ ID No. 102; CDC66 VP2 aa—Vero is SEQ ID No. 108; CDC66 VP 3 aa—Vero is SEQ ID No. 114; CDC66VP 4 aa—Vero is SEQ ID No. 120; CDC66 VP 6 aa—Vero is SEQ ID No. 126;and CDC66 VP 7 aa—Vero is SEQ ID No. 132.

CDC-66 nucleotide sequences of proteins encoded by nucleic acidsisolated at passage 27 isolated from Vero cells: CDC66 NSP1 nt—Vero isSEQ ID No. 69; CDC66 NSP 2 nt—Vero is SEQ ID No. 75; CDC66 NSP 3 nt—Verois SEQ ID No. 81; CDC66 NSP 4 nt—Vero is SEQ ID No. 87; CDC66 NSP 5nt—Vero is SEQ ID No. 93; CDC66 VP1 nt—Vero is SEQ ID No. 99; CDC66 VP 2nt—Vero is SEQ ID No. 105; CDC66 VP 3 nt—Vero is SEQ ID No. 111; CDC66VP 4 nt—Vero is SEQ ID No. 117; CDC66 VP 6 nt—Vero is SEQ ID No. 123;and CDC66 VP 7 nt—Vero is SEQ ID No. 129.

Entire amino acid and nucleotide sequences from CDC-66 rotavirusisolated from stool and Vero cells are compared with entire amino acidand nucleotide sequences of a strain of rotavirus A considered to be theclosest related known rotavirus A, human rotavirus DS-1 strain oranother closely related strain, as shown herein.

As shown in Table 4, CDC-66 genes share high sequence identity with thecorresponding genes of the prototype P[4], G2 strain DS-1.

Table 4. Percentages of nucleotide (NT) and deduced amino acid (AA)identity of rotavirus vaccine strain CDC-66 genome compared with genomesequence of prototype rotavirus strain DS-1.

TABLE 4 Gene nt % aa % VP1 90.85 97.24 VP2 94.11 98.86 VP3 92.9 95.69VP4 94.02 96.52 VP6 87.98 98.74 VP7 93.88 96.32 NSP1 93.12 93.21 NSP286.89 93.99 NSP3 95.31 97.12 NSP4 94.76 95.43 NSP5 92.57 96.5

Changes in nt and aa sequences of entire genome of CDC-66 strain fromstool to the passage in Vero cells have been documented, as shown inTable 5.

TABLE 5 Changes of nt and aa in genes of CDC-66 from stool to passage 27in Vero cells Gene nt change aa change NSP1 none none NSP2 470 C→T 142 H→ Y NSP3 none none NSP4 none none NSP5 126 T→C, 199 A→G 60 I→V VP1 440A→G 141 N→S VP2 none none VP3 760 T→C, 1143 T→C, 365 L→S 1882 A→G VP4770C→A, 1109T→C, 254T→K, 367V→A, 1162G→A, 1184A→G 385D→N, 392E→G VP6none none VP7 none none

Vaccines

Vaccines and methods for their use to induce active immunity andprotection against rotavirus induced illness in a subject are providedaccording to the present invention.

In particular embodiments, vaccine compositions for enhancingimmunological protection against a rotavirus-mediated disease in asubject are provided according to the present invention which includes ahuman rotavirus strain admixed with a pharmaceutically acceptablecarrier.

The term “vaccine composition” is used herein to refers to a compositionincluding a biological agent capable of inducing an immune response in asubject inoculated with the vaccine composition. In particularembodiments, the biological agent is a live attenuated and/or inactiverotavirus. In further embodiments, the biological agent is an antigenicportion of a rotavirus.

In particular embodiments, a human rotavirus strain included in avaccine composition of the present invention is CDC-9 or CDC-66.Combinations of these human rotavirus strains are optionally included invaccine compositions of the present invention. Additionally, a humanrotavirus strain other than CDC-9 or CDC-66 is optionally included in avaccine composition of the present invention.

In particular embodiments of a vaccine composition according to thepresent invention, at least two rotavirus strains are included. The twoor more rotavirus strains each independently have a G1, G2, G3, G4, G5,G6, G7, G8, G9, G10, G11, G12, G13 or G14 G serotype. Thus, for example,at least one of CDC-9 or CDC-66 is present in a vaccine composition ofthe present invention along with at least a second human rotavirusstrain which has a G1, G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12,G13 or G14 G serotype.

Each of the at least two rotavirus strains included in a vaccinecomposition has a P serotype which is P1A, P1B, P2A, P3, P4, P5, P6, P8,P11, or P12 in particular embodiments.

A vaccine composition for enhancing immunological protection against arotavirus-mediated disease in a subject includes a first human rotavirusstrain characterized as having a G1 serotype and a second humanrotavirus strain characterized as having a G2 serotype in particularembodiments of a vaccine composition of the present invention. Each ofthe two rotavirus strains independently has a P group serotype which isP1A, P1B, P2A, P3, P4, P5, P6, P8, P11 or P12.

In some embodiments, the human rotavirus strain having a G1 serotype isCDC-9.

In some embodiments, the human rotavirus strain having a G2 serotype isCDC-66.

Combinations of human rotavirus strains in particular embodiments of avaccine composition of the present invention include CDC-9 and CDC-66.

A human rotavirus strain included in a vaccine composition according tothe present invention is a live attenuated rotavirus or an inactivatedrotavirus. The choice of live attenuated rotavirus or inactivatedrotavirus depends on factors such as route of vaccine compositionadministration.

In a particular embodiment, a vaccine composition including a humanrotavirus A strain CDC-9, and/or CDC-66, one or more rotavirus A CDC-9,and/or CDC-66 polypeptides and/or an immunogenic fragment of one or morerotavirus A CDC-9, and/or CDC-66 polypeptides, stimulates generation ofneutralizing antibodies to a rotavirus A CDC-9, and/or CDC-66 strain.

Vaccine compositions are provided according to embodiments of thepresent invention which include one or more rotavirus A polypeptidesand/or an immunogenic fragment of one or more rotavirus A polypeptides.In particular embodiments of an inventive vaccine composition, a CDC-9,and/or CDC-66 polypeptide, a homolog thereof, and/or an immunogenicfragment thereof is included.

The term “homolog” refers to a polypeptide characterized by amino acidsequence homology to a reference CDC-9 or CDC-66 rotavirus Apolypeptide.

CDC-9 Sequences

Accordingly, the present invention provides a virus including an NSP1having SEQ ID NO: 2 or a homolog having an amino acid sequence that isgreater than 80%, is greater than 81%, greater than 82%, greater than83%, greater than 84%, greater than 85%, greater than 86%, greater than87%, greater than 88%, greater than 89%, greater than 90%, greater than91%, greater than 92%, greater than 93%, greater than 94%, greater than95%, greater than 96%, greater than 97%, greater than 98% or greaterthan 99% identical to SEQ ID NO: 2. Further, the present inventionprovides a virus including an NSP1 having SEQ ID NO: 3 or a homologhaving an amino acid sequence that is greater than 80%, is greater than81%, greater than 82%, greater than 83%, greater than 84%, greater than85%, greater than 86%, greater than 87%, greater than 88%, greater than89%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98% or greater than 99% identical to SEQ ID NO: 3.

The present invention provides a virus including an NSP2 having SEQ IDNO: 5 or a homolog having an amino acid sequence that is greater than80%, is greater than 81%, greater than 82%, greater than 83%, greaterthan 84%, greater than 85%, greater than 86%, greater than 87%, greaterthan 88%, greater than 89%, greater than 90%, greater than 91%, greaterthan 92%, greater than 93%, greater than 94%, greater than 95%, greaterthan 96%, greater than 97%, greater than 98% or greater than 99%identical to SEQ ID NO: 5.

The present invention provides a virus including an NSP3 having SEQ IDNO: 8 or a homolog having an amino acid sequence that is greater than80%, is greater than 81%, greater than 82%, greater than 83%, greaterthan 84%, greater than 85%, greater than 86%, greater than 87%, greaterthan 88%, greater than 89%, greater than 90%, greater than 91%, greaterthan 92%, greater than 93%, greater than 94%, greater than 95%, greaterthan 96%, greater than 97%, greater than 98% or greater than 99%identical to SEQ ID NO: 8.

The present invention provides a virus including an NSP4 having SEQ IDNO: 11 or a homolog having an amino acid sequence that is greater than80%, is greater than 81%, greater than 82%, greater than 83%, greaterthan 84%, greater than 85%, greater than 86%, greater than 87%, greaterthan 88%, greater than 89%, greater than 90%, greater than 91%, greaterthan 92%, greater than 93%, greater than 94%, greater than 95%, greaterthan 96%, greater than 97%, greater than 98% or greater than 99%identical to SEQ ID NO: 11.

The present invention provides a virus including an NSP5 having SEQ IDNO: 14 or a homolog having an amino acid sequence that is greater than80%, is greater than 81%, greater than 82%, greater than 83%, greaterthan 84%, greater than 85%, greater than 86%, greater than 87%, greaterthan 88%, greater than 89%, greater than 90%, greater than 91%, greaterthan 92%, greater than 93%, greater than 94%, greater than 95%, greaterthan 96%, greater than 97%, greater than 98% or greater than 99%identical to SEQ ID NO: 14. Further, the present invention provides avirus including an NSP5 having SEQ ID NO: 15 or a homolog having anamino acid sequence that is greater than 80%, is greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,greater than 90%, greater than 91%, greater than 92%, greater than 93%,greater than 94%, greater than 95%, greater than 96%, greater than 97%,greater than 98% or greater than 99% identical to SEQ ID NO: 15.

Accordingly, the present invention provides a virus including a VP1having SEQ ID NO: 17 or a homolog having an amino acid sequence that isgreater than 80%, is greater than 81%, greater than 82%, greater than83%, greater than 84%, greater than 85%, greater than 86%, greater than87%, greater than 88%, greater than 89%, greater than 90%, greater than91%, greater than 92%, greater than 93%, greater than 94%, greater than95%, greater than 96%, greater than 97%, greater than 98% or greaterthan 99% identical to SEQ ID NO: 17.

The present invention provides a virus including a VP2 having SEQ ID NO:20 or a homolog having an amino acid sequence that is greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,greater than 90%, greater than 91%, greater than 92%, greater than 93%,greater than 94%, greater than 95%, greater than 96%, greater than 97%,greater than 98% or greater than 99% identical to SEQ ID NO: 20.

The present invention provides a virus including a VP3 having SEQ ID NO:23 or a homolog having an amino acid sequence that is greater than 80%,greater than 8%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 23.

The present invention provides a virus including a VP4 having SEQ ID NO:26 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 26. Further, the present invention provides a virus including anVP4 having SEQ ID NO: 27 or a homolog having an amino acid sequence thatis greater than 80%, greater than 81%, greater than 82%, greater than83%, greater than 84%, greater than 85%, greater than 86%, greater than87%, greater than 88%, greater than 89%, greater than 90%, greater than91%, greater than 92%, greater than 93%, greater than 94%, greater than95%, greater than 96%, greater than 97%, greater than 98% or greaterthan 99% identical to SEQ ID NO: 27.

The present invention provides a virus including a VP6 having SEQ ID NO:29 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 29. Further, the present invention provides a virus including anVP6 having SEQ ID NO: 30 or a homolog having an amino acid sequence thatis greater than 80%, greater than 81%, greater than 82%, greater than83%, greater than 84%, greater than 85%, greater than 86%, greater than87%, greater than 88%, greater than 89%, greater than 90%, greater than91%, greater than 92%, greater than 93%, greater than 94%, greater than95%, greater than 96%, greater than 97%, greater than 98% or greaterthan 99% identical to SEQ ID NO: 30.

The present invention provides a virus including a VP7 having SEQ ID NO:32 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 32.

CDC-66 Sequences

The present invention provides a virus including an NSP1 having SEQ IDNO: 71 or a homolog having an amino acid sequence that is greater than80%, greater than 81%, greater than 82%, greater than 83%, greater than84%, greater than 85%, greater than 86%, greater than 87%, greater than88%, greater than 89%, greater than 90%, greater than 91%, greater than92%, greater than 93%, greater than 94%, greater than 95%, greater than96%, greater than 97%, greater than 98% or greater than 99% identical toSEQ ID NO: 71.

The present invention provides a virus including an NSP2 having SEQ IDNO: 77 or a homolog having an amino acid sequence that is greater than80%, greater than 81%, greater than 82%, greater than 83%, greater than84%, greater than 85%, greater than 86%, greater than 87%, greater than88%, greater than 89%, greater than 90%, greater than 91%, greater than92%, greater than 93%, greater than 94%, greater than 95%, greater than96%, greater than 97%, greater than 98% or greater than 99% identical toSEQ ID NO: 77. Further, the present invention provides a virus includingan NSP2 having SEQ ID NO: 78 or a homolog having an amino acid sequencethat is greater than 80%, greater than 81%, greater than 82%, greaterthan 83%, greater than 84%, greater than 85%, greater than 86%, greaterthan 87%, greater than 88%, greater than 89%, greater than 90%, greaterthan 91%, greater than 92%, greater than 93%, greater than 94%, greaterthan 95%, greater than 96%, greater than 97%, greater than 98% orgreater than 99% identical to SEQ ID NO: 78.

The present invention provides a virus including an NSP3 having SEQ IDNO: 83 or a homolog having an amino acid sequence that is greater than80%, greater than 81%, greater than 82%, greater than 83%, greater than84%, greater than 85%, greater than 86%, greater than 87%, greater than88%, greater than 89%, greater than 90%, greater than 91%, greater than92%, greater than 93%, greater than 94%, greater than 95%, greater than96%, greater than 97%, greater than 98% or greater than 99% identical toSEQ ID NO: 83.

The present invention provides a virus including an NSP4 having SEQ IDNO: 89 or a homolog having an amino acid sequence that is greater than80%, greater than 81%, greater than 82%, greater than 83%, greater than84%, greater than 85%, greater than 86%, greater than 87%, greater than88%, greater than 89%, greater than 90%, greater than 91%, greater than92%, greater than 93%, greater than 94%, greater than 95%, greater than96%, greater than 97%, greater than 98% or greater than 99% identical toSEQ ID NO: 89.

The present invention provides a virus including an NSP5 having SEQ IDNO: 95 or a homolog having an amino acid sequence that is greater than80%, greater than 81%, greater than 82%, greater than 83%, greater than84%, greater than 85%, greater than 86%, greater than 87%, greater than88%, greater than 89%, greater than 90%, greater than 91%, greater than92%, greater than 93%, greater than 94%, greater than 95%, greater than96%, greater than 97%, greater than 98% or greater than 99% identical toSEQ ID NO: 95. Further, the present invention provides a virus includingan NSP5 having SEQ ID NO: 96 or a homolog having an amino acid sequencethat is greater than 80%, greater than 81%, greater than 82%, greaterthan 83%, greater than 84%, greater than 85%, greater than 86%, greaterthan 87%, greater than 88%, greater than 89%, greater than 90%, greaterthan 91%, greater than 92%, greater than 93%, greater than 94%, greaterthan 95%, greater than 96%, greater than 97%, greater than 98% orgreater than 99% identical to SEQ ID NO: 96.

Accordingly, the present invention provides a virus including a VP1having SEQ ID NO: 101 or a homolog having an amino acid sequence that isgreater than 80%, greater than 81%, greater than 82%, greater than 83%,greater than 84%, greater than 85%, greater than 86%, greater than 87%,greater than 88%, greater than 89%, greater than 90%, greater than 91%,greater than 92%, greater than 93%, greater than 94%, greater than 95%,greater than 96%, greater than 97%, greater than 98% or greater than 99%identical to SEQ ID NO: 101. Further, the present invention provides avirus including an VP1 having SEQ ID NO: 102 or a homolog having anamino acid sequence that is greater than 80%, greater than 81%, greaterthan 82%, greater than 83%, greater than 84%, greater than 85%, greaterthan 86%, greater than 87%, greater than 88%, greater than 89%, greaterthan 90%, greater than 91%, greater than 92%, greater than 93%, greaterthan 94%, greater than 95%, greater than 96%, greater than 97%, greaterthan 98% or greater than 99% identical to SEQ ID NO: 102.

The present invention provides a virus including a VP2 having SEQ ID NO:107 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 107.

The present invention provides a virus including a VP3 having SEQ ID NO:113 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 113. Further, the present invention provides a virus including anVP3 having SEQ ID NO: 114 or a homolog having an amino acid sequencethat is greater than 80%, greater than 81%, greater than 82%, greaterthan 83%, greater than 84%, greater than 85%, greater than 86%, greaterthan 87%, greater than 88%, greater than 89%, greater than 90%, greaterthan 91%, greater than 92%, greater than 93%, greater than 94%, greaterthan 95%, greater than 96%, greater than 97%, greater than 98% orgreater than 99% identical to SEQ ID NO: 114.

The present invention provides a virus including a VP4 having SEQ ID NO:119 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 119. Further, the present invention provides a virus including anVP4 having SEQ ID NO: 120 or a homolog having an amino acid sequencethat is greater than 80%, greater than 81%, greater than 82%, greaterthan 83%, greater than 84%, greater than 85%, greater than 86%, greaterthan 87%, greater than 88%, greater than 89%, greater than 90%, greaterthan 91%, greater than 92%, greater than 93%, greater than 94%, greaterthan 95%, greater than 96%, greater than 97%, greater than 98% orgreater than 99% identical to SEQ ID NO: 120.

The present invention provides a virus including a VP6 having SEQ ID NO:125 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 125.

The present invention provides a virus including a VP7 having SEQ ID NO:131 or a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 131.

The present invention provides an isolated or purified NSP1, NSP2, NSP3,NSP4, NSP5, VP1, VP2, VP3, VP4, VP6, or VP7. The term “purified” or“isolated” as used herein, is intended to refer to a composition,isolatable from other components, wherein the compositions is purifiedto any degree relative to its naturally-obtainable state, i.e., in thiscase, relative to its purity within a cell, relative to is purity withina virion, or relative to its purity within an infective organism. Anisolated composition, therefore, also refers to a protein, peptide,nucleic acid, or oligonucleotide, substantially free from theenvironment in which it may naturally occur.

It is recognized that numerous variants, analogues, or homologues arewithin the scope of the present invention including amino acidsubstitutions, alterations, modifications, or other amino acid changesthat increase, decrease, or do not alter the function or immunogenicpropensity of the inventive immunogen or vaccine. It is furtherappreciated that the inventive sequences are optionally modified by theaddition of one or more amino acids, sugars, nucleotides, pendentgroups, fluorophores, lumiphores, radioactive molecules, lipids, fattyacids, derivatives thereof, or other groups known in the art.Illustratively, an inventive immunogen is conjugated to a protein.Optionally, an inventive immunogen is conjugated to a protein thatpromotes the immunogenicity of an immunogen, illustratively, keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA), or modificationsthereof, as well as BLUE CARRIER immunogenic protein from ThermoScientific, Rockford, Ill. Other sources of natural or artificialimmunogenic protein conjugates are known in the art. Optionally, aninventive immunogen is conjugated to an antibody. Optionally, aninventive immunogen is conjugated to other regions of G-protein that mayor may not also contain epitopes.

In some embodiments, the NSP1 has SEQ ID NO: 2 or SEQ ID No. 3, or is ahomolog having an amino acid sequence that is greater than 80%, greaterthan 81%, greater than 82%, greater than 83%, greater than 84%, greaterthan 85%, greater than 86%, greater than 87%, greater than 88%, greaterthan 89%, greater than 90%, greater than 91%, greater than 92%, greaterthan 93%, greater than 94%, greater than 95%, greater than 96%, greaterthan 97%, greater than 98% or greater than 99% identical to SEQ ID NO: 2or to SEQ ID No. 3. In a further embodiment, the NSP1 has SEQ ID NO: 71or is a homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 71.

The present invention provides an isolated or purified NSP2. In oneembodiment, the NSP2 has SEQ ID NO: 5 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 5. In a furtherembodiment, the NSP2 has SEQ ID NO: 77 or SEQ ID NO: 78 or is a homologhaving an amino acid sequence that is greater than 80%, greater than81%, greater than 82%, greater than 83%, greater than 84%, greater than85%, greater than 86%, greater than 87%, greater than 88%, greater than89%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98% or greater than 99% identical to SEQ ID NO: 77 orSEQ ID NO: 78.

The present invention provides an isolated or purified NSP3. In oneembodiment, the NSP3 has SEQ ID NO: 8 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 8. In a furtherembodiment, the NSP3 has SEQ ID NO: 83 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 83.

The present invention provides an isolated or purified NSP4. In oneembodiment, the NSP4 has SEQ ID NO: 11 or a homolog having an amino acidsequence that is greater than 80%, greater than 81%, greater than 82%,greater than 83%, greater than 84%, greater than 85%, greater than 86%,greater than 87%, greater than 88%, greater than 89%, greater than 90%,greater than 91%, greater than 92%, greater than 93%, greater than 94%,greater than 95%, greater than 96%, greater than 97%, greater than 98%or greater than 99% identical to SEQ ID NO: 11. In a further embodiment,the NSP4 has SEQ ID NO: 89 or a homolog having an amino acid sequencethat is greater than 80%, greater than 81%, greater than 82%, greaterthan 83%, greater than 84%, greater than 85%, greater than 86%, greaterthan 87%, greater than 88%, greater than 89%, greater than 90%, greaterthan 91%, greater than 92%, greater than 93%, greater than 94%, greaterthan 95%, greater than 96%, greater than 97%, greater than 98% orgreater than 99% identical to SEQ ID NO: 89

The present invention provides an isolated or purified NSP5. In oneembodiment, the NSP5 has SEQ ID NO: 14 or SEQ ID NO. 15 or is a homologhaving an amino acid sequence that is greater than 80%, greater than81%, greater than 82%, greater than 83%, greater than 84%, greater than85%, greater than 86%, greater than 87%, greater than 88%, greater than89%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98% or greater than 99% identical to SEQ ID NO: 14 orSEQ ID NO. 15. In a further embodiment, the NSP5 has SEQ ID NO: 95 orSEQ ID NO: 96 or is a homolog having an amino acid sequence that isgreater than 80%, greater than 81%, greater than 82%, greater than 83%,greater than 84%, greater than 85%, greater than 86%, greater than 87%,greater than 88%, greater than 89%, greater than 90%, greater than 91%,greater than 92%, greater than 93%, greater than 94%, greater than 95%,greater than 96%, greater than 97%, greater than 98% or greater than 99%identical to SEQ ID NO: 95 or SEQ ID NO: 96.

The present invention provides an isolated or purified VP1. In oneembodiment, the VP1 has SEQ ID NO: 17 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 17. In a furtherembodiment, the VP1 has SEQ ID NO: 101 or SEQ ID NO.102 or is a homologhaving an amino acid sequence that is greater than 80%, greater than81%, greater than 82%, greater than 83%, greater than 84%, greater than85%, greater than 86%, greater than 87%, greater than 88%, greater than89%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98% or greater than 99% identical to SEQ ID NO: 101 orSEQ ID NO.102.

The present invention provides an isolated or purified VP2. In oneembodiment, the VP2 has SEQ ID NO: 20 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 20. In a furtherembodiment, the VP2 has SEQ ID NO: 107 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 107.

The present invention provides an isolated or purified VP3. In oneembodiment, the VP3 has SEQ ID NO: 23 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 23. In a furtherembodiment, the VP3 has SEQ ID NO: 113 or SEQ ID NO. 114 or is a homologhaving an amino acid sequence that is greater than 80%, greater than81%, greater than 82%, greater than 83%, greater than 84%, greater than85%, greater than 86%, greater than 87%, greater than 88%, greater than89%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98% or greater than 99% identical to SEQ ID NO: 113 orSEQ ID NO. 114.

The present invention provides an isolated or purified VP4. In oneembodiment, the VP4 has SEQ ID NO: 26 or SEQ ID NO. 27 or is a homologhaving an amino acid sequence that is greater than 80%, greater than81%, greater than 82%, greater than 83%, greater than 84%, greater than85%, greater than 86%, greater than 87%, greater than 88%, greater than89%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98% or greater than 99% identical to SEQ ID NO: 26 orSEQ ID NO. 27. In a further embodiment, the VP4 has SEQ ID NO: 119 orSEQ ID NO. 120 or is a homolog having an amino acid sequence that isgreater than 80%, greater than 81%, greater than 82%, greater than 83%,greater than 84%, greater than 85%, greater than 86%, greater than 87%,greater than 88%, greater than 89%, greater than 90%, greater than 91%,greater than 92%, greater than 93%, greater than 94%, greater than 95%,greater than 96%, greater than 97%, greater than 98% or greater than 99%identical to SEQ ID NO: 119 or SEQ ID NO. 120.

The present invention provides an isolated or purified VP6. In oneembodiment, the VP6 has SEQ ID NO: 29 or SEQ ID NO. 30 or is a homologhaving an amino acid sequence that is greater than 80%, greater than81%, greater than 82%, greater than 83%, greater than 84%, greater than85%, greater than 86%, greater than 87%, greater than 88%, greater than89%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98% or greater than 99% identical to SEQ ID NO: 29 orSEQ ID NO. 30. In a further embodiment, the VP6 has SEQ ID NO: 125 or isa homolog having an amino acid sequence that is greater than 80%,greater than 81%, greater than 82%, greater than 83%, greater than 84%,greater than 85%, greater than 86%, greater than 87%, greater than 88%,greater than 89%, greater than 90%, greater than 91%, greater than 92%,greater than 93%, greater than 94%, greater than 95%, greater than 96%,greater than 97%, greater than 98% or greater than 99% identical to SEQID NO: 125.

The present invention provides an isolated or purified VP7. In oneembodiment, the VP7 has SEQ ID NO: 32 or is a homolog having an aminoacid sequence that is greater than 80%, greater than 81%, greater than82%, greater than 83%, greater than 84%, greater than 85%, greater than86%, greater than 87%, greater than 88%, greater than 89%, greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98% or greater than 99% identical to SEQ ID NO: 32. In a furtherembodiment, the VP7 has SEQ ID NO: 131 or is a homolog having an aminoacid sequence that is greater than 97%, greater than 98% or greater than99% identical to SEQ ID NO: 131.

An isolated or purified nucleic acid encoding an above-described proteinor fragment thereof is provided according to embodiments of the presentinvention. Optionally, the isolated or purified nucleic acid encoding anabove-described protein or fragment thereof is included in a vector.

A nucleic acid encoding NSP1 includes the nucleotide sequence of SEQ IDNO: 35; SEQ ID NO: 36; SEQ ID NO: 68 or a fragment thereof encoding atleast nine contiguous amino acids. A nucleic acid encoding NSP2 includesthe nucleotide sequence of SEQ ID NO: 38; SEQ ID NO: 74; SEQ ID NO: 75;or a fragment thereof encoding at least nine contiguous amino acids. Anucleic acid encoding NSP3 includes the nucleotide sequence of SEQ IDNO: 41; SEQ ID NO: 80; or a fragment thereof encoding at least ninecontiguous amino acids. A nucleic acid encoding NSP4 includes thenucleotide sequence of SEQ ID NO: 44; SEQ ID NO: 86; or a fragmentthereof encoding at least nine contiguous amino acids. A nucleic acidencoding NSP5 includes the nucleotide sequence of SEQ ID NO: 47; SEQ IDNO: 48; SEQ ID NO: 92; SEQ ID NO: 93; or a fragment thereof encoding atleast nine contiguous amino acids. A nucleic acid encoding VP1 includesthe nucleotide sequence of SEQ ID NO: 50; SEQ ID NO: 98; SEQ ID NO: 99;or a fragment thereof encoding at least nine contiguous amino acids. Anucleic acid encoding VP2 includes the nucleotide sequence of SEQ ID NO:53; SEQ NO: 104; or a fragment thereof encoding at least nine contiguousamino acids. A nucleic acid encoding VP3 includes the nucleotidesequence of SEQ ID NO: 23; SEQ ID NO: 110; SEQ ID NO: 111; or a fragmentthereof encoding at least nine contiguous amino acids. A nucleic acidencoding VP4 includes the nucleotide sequence of SEQ ID NO: 59; SEQ IDNO: 60; SEQ ID NO: 116; SEQ ID NO: 117; or a fragment thereof encodingat least nine contiguous amino acids. A nucleic acid encoding VP6includes the nucleotide sequence of SEQ ID NO: 62; SEQ ID NO: 63; SEQ IDNO: 122; or a fragment thereof encoding at least nine contiguous aminoacids. A nucleic acid encoding VP7 includes the nucleotide sequence ofSEQ ID NO: 65; SEQ ID NO: 128; SEQ ID NO: 129; or a fragment thereofencoding at least nine contiguous amino acids.

One of skill in the art will appreciate that, due to the degeneracy ofthe genetic code, a particular polypeptide or fragment thereof can beencoded by more than one nucleic acid sequence.

Mutations can be introduced using standard molecular biology techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis. One ofskill in the art will recognize that one or more amino acid mutationscan be introduced without altering the functional properties ofrotavirus polypeptides. For example, one or more amino acidsubstitutions, additions, or deletions can be made without altering thefunctional properties of rotavirus polypeptides. It is also appreciatedthat several mutations optionally increase, decrease, or do not changethe immunogenicity of an inventive polypeptide.

Conservative amino acid substitutions can be made in rotaviruspolypeptides to produce homologs. Conservative amino acid substitutionsare art recognized substitutions of one amino acid for another aminoacid having similar characteristics. For example, each amino acid may bedescribed as having one or more of the following characteristics:electropositive, electronegative, aliphatic, aromatic, polar,hydrophobic and hydrophilic. A conservative substitution is asubstitution of one amino acid having a specified structural orfunctional characteristic for another amino acid having the samecharacteristic. Acidic amino acids include aspartate, glutamate; basicamino acids include histidine, lysine, arginine; aliphatic amino acidsinclude isoleucine, leucine and valine; aromatic amino acids includephenylalanine, glycine, tyrosine and tryptophan; polar amino acidsinclude aspartate, glutamate, histidine, lysine, asparagine, glutamine,arginine, serine, threonine and tyrosine; and hydrophobic amino acidsinclude alanine, cysteine, phenylalanine, glycine, isoleucine, leucine,methionine, proline, valine and tryptophan; and conservativesubstitutions include substitution among amino acids within each group.Amino acids may also be described in terms of relative size, alanine,cysteine, aspartate, glycine, asparagine, proline, threonine, serine,valine, all typically considered to be small.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionof amino acids whose hydropathic indices are within ±2 is preferred,those within ±1 are particularly preferred, and those within ±0.5 areeven more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly, where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,and 95% sequence identity to the polypeptide of interest.

Rotavirus particles, nucleic acids, polypeptides and fragments thereofcan be produced in recombinant host cells using well-known conventionaltechniques. Any nucleic acid construct, which is effective in producingthe encoded protein or fragment thereof in a host cell, can be used toproduce rotavirus particles, rotavirus polypeptides or fragmentsthereof.

One of ordinary skill in the art recognizes many ways to make theinventive CDC-9 or CDC-66 viruses for use in an inventive vaccinecomposition or in inventive processes. Illustratively, it is commonpractice for one to isolate a putative rotavirus from a stool or otherbiological sample optionally including passaging in cell culture such asin Vero cells similar to the methods illustrated in Esona, M D, et al.,Human Vaccines, 2010; 6:1-7; the contents of which are incorporatedherein by reference. One of skill in the art regularly isolates virusstrains and characterizes the genome sequence by techniques well knownin the art. It is common practice for one of skill in the art tosequence a viral genome and compare the output sequence to a modelsequence such as the sequences of CDC-9 of CDC-66 disclosed herein todetermine whether the isolated virus has the required genetic sequenceto be CDC-9, CDC-66, or homologues thereof.

One of skill in the art also knows methods of modifying a modelrotavirus such as KU or DS-1 to make CDC-9 or CDC-66 viruses. One suchmethod uses the reverse genetics approach of Komoto, S., et al., PNASUSA, 2006; 103:4646-4651, the contents of which are incorporated hereinby reference. Briefly, each of the genes of strain KU can be isolatedand amplified by taking culture fluid from infected MA104 cells,extracting the viral dsRNA and synthesizing cDNA using with avianmyeloblastosis virus reverse transcriptase (Seikagaku Kogyo, Tokyo,Japan) using a starting primer. It is well within the level of skill inthe art to design primers for synthesizing cDNA. Numerous free andcommercially available programs for primer synthesis are known to thoseof skill in the art. Illustratively, primers for the KU VP4 gene aredescribed in Komoto, S., et al., PNAS USA, 2006; 103:4646-4651.Modification of the sequences of KU or any other strain to those ofCDC-9 or CDC-66 are illustratively accomplished by establishing thesequence modifications such as by sequence alignments. Once thenucleotide substitutions are elucidated, modifications of the cDNA canbe achieved by using the QUICKCHANGE XL site-directed mutagenesis kitavailable from Agilent Technologies, Santa Clara, Calif. Modified genesequences to conform to that of CDC-9, CDC-66, or homologues thereof areoptionally inserted into a cell line such as COS-7 cells along with ahelper virus such as KU used to serve as a base for gene insertion intonew viruses. The modified viruses are subsequently isolated by knowntechniques. An optional iterative process is used whereby eachindividual gene of CDC-9 or CDC-66 is substituted for the gene of thehelper virus step wise whereby the isolated substituted virus strainfrom the substitution of the first gene is used as a helper virus forsubstitution of the second gene and so on until CDC-9 or CDC-66 iscreated from a source strain.

Illustrative examples of helper viruses or model rotaviruses can befound at GenBank Accession Nos: (a) VP3 strains: RV161-00 (DQ490547),RV176-00 (DQ490553), DRC88 (DQ005112), DRC86 (DQ005123), TB-Chen(AY787654), DS-1 (AY277914), AU-1 (DQ490537), T152 (DQ146701), Hosokawa(DQ870491), Hochi (AY277915), Wa (AY267335), L26 (AY277918), KU(AB022767), Dhaka25-02 (DQ146651), Dhaka12-03 (DQ146662), B4633-03(DQ146640), PO-13 (AB009631).

(b) VP7 (G1) strains: SK417 (EU839907), DH415 (EU839906), MMC82(EU839913), Dhaka18-02 (AY631051), MMC56 (EU839911), Matlab159-02(AY631055), ISO-4 (AY098670), Thai-2104 (DQ512982), CMH042/04(EF199713), 417 (D16328), T73 (AF450291), TE1 (D17721), K18 (D16319),Chi-87 (DQ512998), J-4825 (DQ512989), 88H249 (AB081795), 421 (D16326),RV-4 (M64666), AU007 (AB081799), HOU8697 (U88717), Mvd9607 (AF480295),80 (D16325), DC03 (AF183859), KU (AB222788), K2 (D16323), K8 (D16344),Egy-8 (U26374), Brz-5 (U26367), Wa (K02033), D (AB118022), C95 (L24165),T449 (M92651), DS-1 (AB118023).

(c) VP4 P[8] strains: ITO (AB008280), D (EF672570), Wa (L34161), Hochi(AB008295), Odelia (AB008296), VA70 (AJ540229), CH32 (AB008274), MO(AB008278), KU (AB222784), Wi61 (EF672619), F45 (U30716) P (EF672598),AI-39 (AB008283), 90-544 (AB008304), B4633-03 (DQ146641), Dhaka25-02(DQ146652), SK438 (EU839955), DH402 (EU839958), DH415 (EU839955), DS-1(AB118025).

(d) NSP4 strains: Dhaka16-03 (DQ492678), 1099 (AJ236759), Dhaka12-03(DQ146669), Dhaka25-02 (DQ146658), KU (AB022772), Wa (AF093199), RMC321(AF541921), OSU (D88831), AU-1 (D89873), CMH120/04 (DQ923799), B4106(AY740732), C-11 (AF144793), DRC86 (DQ005116), DRC88 (DQ005105), DS-1(AF174305), TB-Chen (AY787650), Ch-1 (AB065287).

Each of the files and sequences at the aforementioned accession numbersare incorporated herein by reference.

Other methods, primers, isolation techniques, sequencing techniques, andcharacterization techniques are known to those of skill in the art andare similarly operable herein. Illustratively, one can reconstituteCDC-9 or CDC-66 viruses de novo from isolated genes such as by assemblyof virus particles with captured genes illustratively by the techniquesof or modifications of Gonzalez, S A, and Affranchino, J L, J. Gen.Virol., 1995; 76:2357-2360, the contents of which are incorporatedherein by reference.

Expression constructs and methods for their generation and use toexpress a desired protein are known in the art, as described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2001; Ausubel, F. et al., (Eds.),Short Protocols in Molecular Biology, Wiley, 2002; and S. J. Higgins andB. D. Hames (Eds.), Protein Expression: A Practical Approach, OxfordUniversity Press, USA, 1999.

Illustratively, a nucleic acid molecule encoding one or more rotaviruspolypeptides and/or rotavirus polypeptide fragments is operably linkedto regulatory sequences that control transcriptional expression in anexpression vector. The expression vector is introduced into a host celland the produced rotavirus particles, one or more rotavirus polypeptidesand/or rotavirus polypeptide fragments can then be isolated.Illustrative constructs include operably linking a rotavirus nucleicacid molecule into plasmid pT7 by first amplifying the nucleic acidmolecule using primers including a T7 promoter sequence and ligating theamplified nucleic acids into the T7 expression vector pX8dT as describedby Schnell, M J, et al., EMBO J, 1994; 13:4195-4203, the contents ofwhich are incorporated herein by reference.

Non-limiting examples of regulatory sequences that controltranscriptional expression in an expression vector illustrativelyinclude a promoter, an enhancer, a splicing signal, a transcriptionstart site, a transcription termination signal, a polyadenylationsignal, an internal ribosome entry site (IRES) and combinations of theseor other regulatory sequences.

Expression vectors include, but are not limited to, viral vectors andbacterial vectors used to express a desired protein. Non-limitingexamples of expression vectors include bacterial plasmids,bacteriophage, adenovirus, adeno-associated virus, herpes virus,vaccinia virus and lentivirus.

A host cell for expression of polypeptides and fragments thereof can beprokaryotic or eukaryotic, such as bacterial, plant, insect, fungus,yeast, and mammalian cells.

An expression vector is introduced into a host cell using well-knowntechniques such as infection or transfection, including calciumphosphate transfection, liposome-mediated transfection, electroporationand sonoporation.

In addition to recombinant methodology, chemical synthetic techniquescan be used to produce polypeptides and fragments thereof. For example,solid phase synthesis, solution phase synthesis, partial solid phasesynthesis or fragment condensation can be used.

The term “isolated” as used herein refers to a substance that has beenseparated from other cellular components associated with the substancein nature or when recombinantly produced not intended to be associatedwith the substance and that may interfere with use of the substance intherapeutic, prophylactic, diagnostic, research or other uses.Generally, an isolated substance described herein is at least about 80%pure, at least about 90% pure, at least about 95% pure, or greater thanabout 99% pure. Purification is achieved using well-known standardmethodology such as fractionation and/or chromatography, such asammonium sulfate precipitation and elution chromatography such as sizeexclusion chromatography, displacement chromatography, ion exchangechromatography and bioaffinity chromatography. Exemplary purificationmethodology is described in S. Doonan, Protein Purification ProtocolsHumana Press, 1996.

Percent identity is determined by comparison of amino acid or nucleicacid sequences, including a reference rotavirus A amino acid or nucleicacid sequence and a putative homologue amino acid or nucleic acidsequence. Algorithms used for determination of percent identityillustratively include the algorithms of S. Karlin and S. Altshul, PNAS,90:5873-5877, 1993; T. Smith and M. Waterman, Adv. Appl. Math.2:482-489, 1981, S. Needleman and C. Wunsch, J. Mol. Biol., 48:443-453,1970, W. Pearson and D. Lipman, PNAS, 85:2444-2448, 1988 and othersincorporated into computerized implementations such as, but not limitedto, GAP, BESTFIT, FASTA, TFASTA; and BLAST, for example incorporated inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis. and publicly available from the NationalCenter for Biotechnology Information.

Rotavirus A CDC-9, and/or CDC-66 polypeptides, a homolog thereof, and/oran immunogenic fragment thereof may be prepared by any of variousmethods, including isolation of virus particles from sources such ascultured cells or patient samples, isolation of one or more polypeptidesand/or one or more polypeptide fragments from viral particles,recombinant production of viral polypeptides, fragments and/or viralparticles, including intact and virus-like particles, and/or by chemicalsynthetic techniques. Methods of isolation of virus particles, viruspolypeptides and/or one or more virus polypeptide fragments, recombinantproduction of virus polypeptides, virus polypeptide fragments and/orvirus particles are described in detail herein, in references citedherein, and are known to those of skill in the art.

An antigen may be made more immunogenic if desired by linkage to acarrier molecule such bovine serum albumin or keyhole limpet her ocyaninand/or by use of an adjuvant. Carrier linkage may be accomplished by anyof various techniques, illustratively including, but not limited to,conjugation and expression of a fusion protein.

Recombinantly expressed polypeptides or peptides may be tagged to allowfor easier isolation. For instance, such polypeptides and peptides areoptionally tagged illustratively, Fc-tagged, 6×HIS-tagged, FLAG-tagged,or by other tag suitable for isolation of a tagged polypeptide.

Vaccine Formulation

In particular embodiments of the invention, a rotavirus A strain forinclusion in a vaccine composition of the present invention is preparedby standard methods typically used for preparation of live orinactivated rotavirus. For example, generally a compatible cell type isinoculated with a rotavirus strain and the cells are maintained underconditions which allow for viral replication and production ofinfectious particles.

A particular example of a cell type which permits rotavirus infection,replication and particle production is a mammalian cell line such as aVero cell line.

Rotavirus particles are harvested, typically from cell culturesupernatant for inclusion in a vaccine composition. The rotavirusparticles may be isolated from the cell culture supernatant, for exampleby filtration and/or centrifugation. The isolated rotavirus particlesare optionally lyophilized, such as for later resuspension in apharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” refers to a carrier whichis substantially non-toxic to a subject and substantially inert to therotavirus included in a vaccine composition. A pharmaceuticallyacceptable carrier is a solid, liquid or gel in form and is typicallysterile and pyrogen free.

A vaccine composition of the present invention may be in any formsuitable for administration to a subject.

A vaccine composition is administered by any suitable route ofadministration including oral and parenteral such as intravenous,intradermal, intramuscular, mucosal, nasal, or subcutaneous routes ofadministration.

For example, a vaccine composition for parenteral administration may beformulated as an injectable liquid including a rotavirus and apharmaceutically acceptable carrier. Examples of suitable aqueous andnonaqueous carriers include water, ethanol, polyols such as propyleneglycol, polyethylene glycol, glycerol, and the like, suitable mixturesthereof; vegetable oils such as olive oil; and injectable organic esterssuch as ethyloleate. Proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of a desirableparticle size in the case of dispersions, and/or by the use of asurfactant, such as sodium lauryl sulfate. A stabilizer is optionallyincluded such as, for example, sucrose, EDTA, EGTA, and an antioxidant.

A solid dosage form for administration or for suspension in a liquidprior to administration illustratively includes capsules, tablets,powders, and granules. In such solid dosage forms, a rotavirus isadmixed with at least one carrier illustratively including a buffer suchas, for example, sodium citrate or an alkali metal phosphateillustratively including sodium phosphates, potassium phosphates andcalcium phosphates; a filler such as, for example, starch, lactose,sucrose, glucose, mannitol, and silicic acid; a binder such as, forexample, carboxymethylcellulose, alignates, gelatin,polyvinylpyrrolidone, sucrose, and acacia; a humectant such as, forexample, glycerol; a disintegrating agent such as, for example,agar-agar, calcium carbonate, plant starches such as potato or tapiocastarch, alginic acid, certain complex silicates, and sodium carbonate; asolution retarder such as, for example, paraffin; an absorptionaccelerator such as, for example, a quaternary ammonium compound; awetting agent such as, for example, cetyl alcohol, glycerolmonostearate, and a glycol; an adsorbent such as, for example, kaolinand bentonite; a lubricant such as, for example, talc, calcium stearate,magnesium stearate, a solid polyethylene glycol or sodium laurylsulfate; a preservative such as an antibacterial agent and an antifungalagent, including for example, sorbic acid, gentamycin and phenol; and astabilizer such as, for example, sucrose, EDTA, EGTA, and anantioxidant.

Solid dosage forms optionally include a coating such as an entericcoating. The enteric coating is typically a polymeric material.Preferred enteric coating materials have the characteristics of beingbiocrodible, gradually hydrolyzable and/or gradually water-solublepolymers. The amount of coating material applied to a solid dosagegenerally dictates the time interval between ingestion and drug release.A coating is applied having a thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below 3associated with stomach acids, yet dissolves above pH 3 in the smallintestine environment. It is expected that any anionic polymerexhibiting a pH-dependent solubility profile is readily used as anenteric coating in the practice of the present invention to achievedelivery of the active agent to the lower gastrointestinal tract. Theselection of the specific enteric coating material depends on propertiessuch as resistance to disintegration in the stomach; impermeability togastric fluids and active agent diffusion while in the stomach; abilityto dissipate at the target intestine site; physical and chemicalstability during storage; non-toxicity; and ease of application.

Suitable enteric coating materials illustratively include cellulosicpolymers such as hydroxypropyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose,cellulose acetate, cellulose acetate phthalate, cellulose acetatetrimellitate, hydroxypropylmethyl cellulose phthalate,hydroxypropylmethyl cellulose succinate and carboxymethylcellulosesodium; acrylic acid polymers and copolymers, preferably formed fromacrylic acid, methacrylic acid, methyl acrylate, ammoniummethylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl; vinylpolymers and copolymers such as polyvinyl pyrrolidone, polyvinylacetate, polyvinylacetate phthalate, vinylacetate crotonic acidcopolymer, and ethylene-vinyl acetate copolymers; shellac; andcombinations thereof. A particular enteric coating material includesacrylic acid polymers and copolymers described for example U.S. Pat. No.6,136,345.

The enteric coating optionally contains a plasticizer to prevent theformation of pores and cracks that allow the penetration of the gastricfluids into the solid dosage form. Suitable plasticizers illustrativelyinclude, triethyl citrate (Citroflex 2), triacetin (glyceryltriacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400(polyethylene glycol 400) diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, a coating composed of ananionic carboxylic acrylic polymer typically contains approximately 10%to 25% by weight of a plasticizer, particularly dibutyl phthalate,polyethylene glycol, triethyl citrate and triacetin. The coating canalso contain other coating excipients such as detackifiers, antifoamingagents, lubricants (e.g., magnesium stearate), and stabilizers (e.g.hydroxypropylceliulose, acids or bases) to solubilize or disperse thecoating material, and to improve coating performance and the coatedproduct.

Liquid dosage forms for oral administration include rotavirus and apharmaceutically acceptable carrier formulated as an emulsion, solution,suspension, syrup, or elixir. A liquid dosage form of a vaccinecomposition of the present invention may include a wetting agent, anemulsifying agent, a suspending agent, a sweetener, a flavoring, or aperfuming agent.

Detailed information concerning customary ingredients, equipment andprocesses for preparing dosage forms is found in Pharmaceutical DosageForms: Tablets, eds. H. A. Lieberman et al., New York: Marcel Dekker,Inc., 1989; and in L. V. Allen, Jr. et al., Ansel's PharmaceuticalDosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, Pa.:Lippincott, Williams & Wilkins, 2004, throughout and in chapter 16; A.R. Gennaro, Remington: The Science and Practice of Pharmacy, LippincottWilliams & Wilkins, 21^(st) ed., 2005, particularly chapter 89; and J.G. Hardman et al., Goodman & Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill Professional, 10th ed., 2001.

An adjuvant is optionally included in a virus composition according toembodiments of the present invention. Adjuvants are known in the art andillustratively include Freund's adjuvant, aluminum hydroxide, aluminumphosphate, aluminum oxide, saponin, dextrans such as DEAE-dextran,vegetable oils such as peanut oil, olive oil, and/or vitamin E acetate,mineral oil, bacterial lipopolysaccharides, peptidoglycans, andproteoglycans.

The term “subject” is used herein to refer to a human. Non-humananimals, illustratively including other primates, cows, horses, sheep,goats, pigs, dogs, cats, birds, poultry, and rodents, are also referredto by the term subject in particular embodiments of the presentinvention.

The isolated rotavirus is treated to inactivate or attenuate therotavirus. Thus, in particular embodiments a vaccine for human rotavirusincludes a live attenuated human rotavirus or an inactivated humanrotavirus.

The term “live attenuated rotavirus” refers to a rotavirus having theability to infect an appropriate host or host cell and replicate and theterm is used to distinguish an “inactivated” rotavirus. The term “liveattenuated rotavirus” refers to a rotavirus characterized bysubstantially diminished virulence compared to wild type humanrotavirus. The term “virulence” is used to describe the degree ofpathogenicity of a rotavirus to a host cell or a host organism.Virulence is determined using any of various assays recognized in theart. For example, virulence may be assessed by exposing cultured hostcells to an attenuated rotavirus and determining the number of cellswhich display a pathogenic response and/or the severity of pathogenicresponse elicited. Diminished virulence is present where an attenuatedrotavirus has decreased capability to cause one or more pathogeniceffects in a host cell and/or host organism.

The term “inactivated” rotavirus is used herein to refer to rotavirusthat has been killed and which is therefore capable of neitherreplication nor infection of a host cell or host organism.

Inactivation is achieved by any of various techniques illustrativelyincluding inactivation using one or more chemical agents, thermalinactivation and/or UV light inactivation.

Chemical agents used to inactivate a rotavirus are known in the art andinclude such agents as ethyleneimines such as binary ethyleneimine;cross-linking aldehydes such as formaldehyde and glutaraldehyde;proteases illustratively including pronase, trypsin and/or chymotrypsin;and detergents such as octylphenol ethoxylates and alkyltrimethylammonium salts. Rotavirus may be inactivated by treatment witha base, for example by incubation of the rotavirus at a pH above pH10.0.

Thermal inactivation may be achieved by heating at temperatures above50° centigrade, for example.

Inactivation is assessed by techniques standard in the art,illustratively including sampling virus at various times during aninactivation procedure and observing cytopathic effects or infectivityof a sample on suitable cells, such as Vero cells.

It is appreciated that, in addition to live attenuated and inactivatedrotavirus, an antigenic portion of a rotavirus is optionally included ina vaccine composition of the present invention. Thus, for example, arotavirus-derived protein or peptide capable of inducing animmunological response in a subject is considered within the scope ofthe present invention. In particular, an antigenic portion of a humanrotavirus strain identified as CDC-9 or CDC-66 is optionally included ina vaccine composition of the present invention.

Methods of inducing an immunological response against arotavirus-mediated disease in a subject are provided according toembodiments of the present invention which include administering atherapeutic amount of a vaccine composition including at least one humanrotavirus strain.

The phrase “therapeutically effective amount” is used herein to refer toan amount effective to induce an immunological response sufficient toprevent or ameliorate signs or symptoms of a rotavirus-mediated disease.Induction of an immunological response in a subject can be determined byany of various techniques known in the art, illustratively includingdetection of anti-rotavirus antibodies, measurement of anti-rotavirusantibody titer and/or lymphocyte proliferation assay. Illustrativemethods for detection of anti-rotavirus antibodies are illustrated byTsunemitsu, H, et al., J. Clin. Microbiol., 1992; 30:2129-2134, thecontents of which are incorporated herein by reference. Signs andsymptoms of rotavirus-mediated disease may be monitored to detectinduction of an immunological response to administration of a vaccinecomposition of the present invention in a subject. An immunologicalresponse is illustratively a reduction of clinical signs and symptoms ofrotavirus-mediated disease such as reduction of the amount of virus shedin feces, reduction of the number of days on which virus is shed infeces, reduction in the number of days the subject has diarrhea,reduction in mortality, reduction in morbidity, reduction in weight lossor weight gain. An immunological response is illustratively, developmentof anti-rotavirus antibodies, activation of T-cells, B-cells, or otherimmune cells following administration of an inventive composition, orother immune responses known in the art.

In a particular embodiment, a method of inducing an immunologicalresponse against a rotavirus-mediated disease in a subject includesadministering 10⁴ to 10⁸ ffu of live attenuated vaccine or 1 to 25micrograms of inactivated virus in a typical vaccine composition.

In some embodiments, a method of inducing an immunological responseagainst a rotavirus-mediated disease in a subject includes administeringa therapeutically effective amount of a vaccine composition including ahuman rotavirus strain CDC-9 and/or CDC-66, polypeptide fragmentsthereof, homologues thereof, or combinations thereof.

In a further embodiment, a method of inducing an immunological responseagainst a rotavirus-mediated disease in a subject includes administeringa therapeutically effective amount of a vaccine composition including aG1 group serotype and a second human rotavirus strain characterized ashaving a G2 group serotype.

Administration of a vaccine composition according to a method of thepresent invention includes administration of one or more doses of avaccine composition to a subject at one time in particular embodiments.Alternatively, two or more doses of a vaccine composition areadministered at time intervals of days, weeks, or years. A suitableschedule for administration of vaccine composition doses depends onseveral factors including age and health status of the subject, type ofvaccine composition used and route of administration, for example. Oneof skill in the art is able to readily determine a dose and schedule ofadministration to be administered to a particular subject.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Example 1—Adaptation and Passaging

One ml of a 10% virus suspension in DMEM is supplemented with neomycinin a 1.7 ml sterile low bind tube, mixed well and then centrifuged for10 min at 3,000 rpm in an Eppendorf microcentrifuge. The supernatant istransferred to a new tube and centrifuged for 10 min at 10,000 rpm(8,000×g). The clarified supernatant is sterilized by passing though a0.45 micron pore filter. The supernatant is tested by EIA (Rotaclone;Meridian Biosciences) and if OD value is >1.0, store it at 4° C. beforeuse for infection. Stool extraction and Rotaclone testing can be donethe day before infection.

The culture medium is removed from cell monolayers in individual rollertubes. Each roller tube is washed with 2 ml of maintenance medium, then2 ml maintenance medium is added to each tube and incubated at 37° C. ina rolling apparatus until virus inoculum is ready.

An aliquot of 0.5 ml of supernatant is transferred to a sterile tube and1 microliter of CaCl₂ stock (300 grams per liter) is added to make afinal concentration of 800 micrograms per milliliter. The tube isincubated at room temperature for 30 min before adding 3 microliters ofporcine trypsin stock (2.5 milligrams per milliliter)—finalconcentration of 15 micrograms per milliliter. The mixture is incubatedfor 60 min at 37° C. The same volume of MEM is treated in the same wayas a mock inoculum. Separate pipette tips are used for pipetting virussuspension and trypsin solutions. All pipetting of virus should be donewithin the biological safety cabinet.

Medium is removed from each roller tube and 0.2 to 0.3 milliliter oftrypsinized virus suspension or mock inoculums is added to each rollertube using separate sterile pipette. The caps are tightened and thetubes incubated at 37° C. on a roller tube apparatus located in anincubator. After 2 hrs incubation, inoculum is removed using a 1 mlpipette and washed gently with 2 ml maintenance medium.

Two milliliters of maintenance medium containing various concentrations(10, 20 or 30 micrograms per ml depending on strain) of trypsin is addedinto each tube and incubated for 2 hours at 37° C. on a roller tubeapparatus located in an incubator.

The cells are observed daily for cytopathic effect (CPE), harvested atday 4 and stored at −70° C. The cells are subjected to freeze-thaw twotimes before the next passage.

The freeze-thawed cell lysates are treated with CaCl₂ and trypsin asdescribed above and subsequent passages are performed as above. Thecells are subjected to freeze-thaw at least once and assayed forrotavirus antigen by Rotaclone kit or virus titer is determined by FFAassays.

Example 2—Production and Purification of Rotavirus Strains

Production of rotavirus is accomplished by use of large scale productionroller bottles. Briefly described, Vero cells are cultured in Dulbecco'sModified Eagle Medium supplemented with 5% fetal bovine serum(Invitrogen Corp., Grand Island, N.Y.) and 50 micrograms/milliliter ofneomycin (Sigma Corp., St. Louis, Mo.). Confluent monolayers of Verocells in roller bottles are infected with a particular rotavirus strainat a multiplicity of infection of 0.1.

Rotavirus obtained by large scale production is purified according tostandard operating procedures. Briefly described, rotavirus is harvestedfrom infected cultures of Vero cells at four days post-infection.Triple-layered rotavirus particles are purified from supernatants bycentrifugation through 40% sucrose cushions in TNC buffer for 2 hours at106,750×g using an SW32Ti rotor and then through isopycniccentrifugations in CsCl gradients for 17 hours at 111,160 g using anSW40Ti rotor. Rotavirus particles may also be purified using sucrosegradients. TNC buffer is 10 mM Tris, pH 8.0, 140 mM NaCl, and 10 mMCaCl₂. Purified rotavirus particles are resuspended in diluent bufferwhich is Hanks Balanced Salt Solution with CaCl₂ and MgCl₂, obtainedfrom Invitrogen Corp., Grand Island, N.Y., supplemented with 10%sorbitol (Sigma Corp., St. Louis, Mo.). The resuspended isolatedrotavirus is stored at −70° C. until it is inactivated and administeredto a subject in this example.

Purified rotavirus is analyzed for purity and identity by any of varioustechniques, illustratively including SDS-PAGE followed by Coomassie bluestaining, western blot using a rotavirus-specific antibody and/orelectron microscopy. In addition, purity and identity of purifiedrotavirus strains is accomplished by identification of particularstructural viral proteins.

FIG. 4A shows CsCl purified rotavirus of strain CDC-9. FIG. 4B showsidentified structural viral proteins of double- and triple-layered CDC-9analyzed by SDS-PAGE in comparison to molecular weight markers.

Example 3—Immunogenicity of Inactivated Rotavirus (IRV)

Immunogenicity of rotavirus strains is tested in a mouse model. Purifiedkilled rotavirus particles are administered intramuscularly to micewithout an adjuvant. Animals are immunized with amounts of killedrotavirus protein in the range between 2 and 20 micrograms.

Immunogenicity is assayed by measuring immunoglobulin titers includingIgM, IgA and IgG in blood samples obtained at various times afteradministration. Neutralizing antibody titers are measured bymicroneutralization assay as described in detail in Jiang, B., Vaccine,17:1005-1013, 1999, the contents of which are incorporated herein byreference. Briefly described, mouse sera are serially diluted two-foldin duplicate wells and incubated with trypsin-inactivated RRV rotavirus.Activated rotavirus or similarly treated serum-free MEM medium isincubated in the absence of mouse serum and serve as positive andnegative controls, respectively. MA104 cells in MEM medium supplementedwith a final concentration of 10 micrograms/milliliter trypsin and 0.5%chick serum, obtained from Invitrogen Corp., Grand Island, N.Y., areadded to each well. After incubation at 37° C. for 18 hours, cells arefixed with formalin. Rotavirus antigens in MA104 cells are detected byincubating cells with rabbit anti-RRV hyperimmune serum, HRP-labeledanti-rabbit IgG, and then tetramethyl benzidine. Neutralizing antibodytiter in a serum is defined as the reciprocal of the highest dilutiongiving a 70% reduction in absorbance value compared to that in the viruscontrol.

Antibody titers in mice injected with killed purified rotavirusparticles are compared with antibody titers in control mice. Antibodytiters in control mice are typically less than 100. FIGS. 5A and 5Billustrate total antibody and neutralizing antibody titers in controland vaccinated mice. Total serum antibody (FIG. 5A) and neutralizingantibody (FIG. 5B) responses to thermally inactivated rotavirus in mice.Mice are vaccinated I.M. twice with killed YK-1 and rotavirus-specifictotal (IgA, IgG, and IgM) and neutralizing antibodies are determined byEIA as described. For total antibody, each serum specimen is tested atan initial dilution of 1:100. Pre-bleed serum specimens have nodetectable antibody at this dilution. A value of 20 is used fordetermining geometric mean titers and illustration. Neutralizingantibody is tested at an initial dilution of 1:20. Antibody titers areexpressed as the geometric means for each group (n=7 or 6). Error barsrepresent 1 standard error.

Example 4—Adjuvant

In a further example, Al(OH)₃ is added as an adjutant to rotavirusparticles in a vaccine administered to mice. Animals are immunizedintramuscularly once with 2 micrograms or 0.2 micrograms of killedpurified rotavirus particles in the presence or absence of 600micrograms Al(OH)₃. AlOH dramatically enhances total antibody titers inmice at both concentrations of rotavirus administered. No antibodytiters (less than 100 dilutions) are detected in control mice immunizedwith 600 micrograms of Al(OH)₃.

FIG. 6 is a bar graph showing total serum antibody responses tothermally inactivated rotavirus formulated with Al(OH)₃ in control andvaccinated mice. Mice are vaccinated I.M. once with killed YK-1 in theabsence or presence of Al(OH)₃ and rotavirus-specific total (IgA, IgG,and IgM) antibodies are determined by EIA as described. For totalantibody, each serum specimen is tested at an initial dilution of 1:100.Pre-bleed serum specimens have no detectable antibody at this dilution.A value of 20 is used for determining geometric mean titers andillustration. Antibody titers are expressed as the geometric means foreach group (n=6). Error bars represent 1 standard error.

Example 5—Gnotobiotic Piglet Model

A gnotobiotic piglet model of rotavirus disease is used. This pigletmodel allows testing under defined conditions avoiding problems ofenvironment exposure of animals and using disease as the outcomevariable. This model also allows testing of an inactivated rotavirusvaccine having a G1 serotype against a homotypic Wa challenge.Gnotobiotic piglets are the best current animal model for infection anddisease with human rotavirus strains. (See Saif L J, et al., Archives ofVirology, 1996; 12:S153-61; and Iosef C, et al., Vaccine, 2002;20:1741-53; the contents of each of which are incorporated herein byreference.)

Thirteen infant gnotobiotic piglets are selected and randomly assignedto 4 groups as indicated in Table 6.

TABLE 6 Piglet Study Design Number of Piglets CDC-9 Antigen Aluminumphosphate Group Name in Group (micrograms) (micrograms) AA 4 0 750 BB 475 0 CC 3 75 750 DD 2 0 (buffer) 0 (buffer)

Each group of animals indicated in Table 6 is kept in separateisolators. Animals in groups BB and CC are vaccinated intramuscularly 3times with an inactivated rotavirus vaccine without or with an adjuvant,respectively. The vaccine formulation in this example includes 75micrograms of killed purified CDC-9 rotavirus in diluent mixed with 750micrograms of aluminum phosphate. Animals in groups AA and DD arevaccinated with 750 micrograms of aluminum phosphate and buffer,respectively, in the same manner. Antigen adsorption is determined bythe Bradford method which showed that about 50% of the antigen was boundto aluminum phosphate. Both bound and unbound antigen was injected inthese immunizations.

As shown in Table 6, piglets are immunized with a vaccine formulationincluding no antigen and 750 micrograms of aluminum phosphate; 75micrograms of antigen and no aluminum phosphate; 75 micrograms ofantigen and 750 micrograms of aluminum phosphate; or no antigen and noaluminum phosphate, that is buffer alone. Each vaccination is carriedout by injecting 0.5 milliliters of the vaccine formulation into musclesof the hind legs of the piglets. After 3 doses of the vaccineformulation administered at intervals of 10-12 days, piglets are orallychallenged with virulent Wa rotavirus. Prior to virus challenge, eachpiglet is inoculated with 3 milliliters of sodium bicarbonate toneutralize acids in the stomach. Fecal specimens are collected from thechallenged piglets daily for 10 days. Blood samples are collectedthroughout the experiment at intervals of 7-14 days. FIG. 7A shows virusshedding in fecal samples of piglets vaccinated with no antigen and with750 micrograms of aluminum phosphate in 4 animals. FIG. 7B shows virusshedding in fecal samples from piglets immunized with antigen and noadjuvant. FIG. 7C shows virus shedding in fecal samples of pigletsimmunized with antigen and adjuvant. FIG. 7D shows virus sheddingmeasured in fecal samples of piglets immunized with buffer only. Theseillustrate that piglets that are mock vaccinated with aluminum phosphateonly or diluent buffer only all shed rotavirus up to 5 days and at hightiter. By contrast, piglets that are vaccinated with inactivatedrotavirus without aluminum phosphate are partially protected, asevidenced by a shortened 1-day shedding or a delayed and reducedshedding. Of the 3 piglets that are vaccinated with inactivatedrotavirus and aluminum phosphate, 2 are completely protected and 1 hasonly a short, 1-day, reduced shedding. Thus, these results showeffectiveness of vaccine formulation according to embodiments of thepresent invention.

Example 6—Gnotobiotic Piglet Model—II

To repeat the experiment above, eleven infant gnotobiotic piglets areselected and randomly assigned to 2 groups as indicated in Table 7.

TABLE 7 Piglet Study Design Number of Piglets CDC-9 Antigen aluminumphosphate Group Name in Group (micrograms) (micrograms) GG 5 0 600 HH 650 600

As shown in Table 7, and as described by Wang, Y, et al., Inactivatedrotavirus vaccine induces protective immunity in gnotobiotic piglets, inpress, the contents of which are incorporated herein by reference,piglets are immunized with a vaccine formulation including no antigenand 600 micrograms of aluminum phosphate or 50 micrograms of antigen and600 micrograms of aluminum phosphate. Each vaccination is carried out byinjecting 0.5 milliliters of the vaccine formulation into muscles of thehind legs of the piglets. After 3 doses of the vaccine formulationadministered at intervals of 10-12 days, piglets are orally challengedwith virulent Wa rotavirus. Prior to virus challenge, each piglet isinoculated with 3 milliliters of sodium bicarbonate to neutralize acidsin the stomach. Fecal specimens are collected from the challengedpiglets daily for 10 days. Blood samples are collected throughout theexperiment at intervals of 7-14 days.

FIG. 8A shows the level of rotavirus specific IgG antibody response insera of piglets vaccinated with no antigen and with 600 micrograms ofaluminum phosphate (solid bars) or piglets vaccinated with 50 microgramsof antigen and with 600 micrograms of aluminum phosphate (hatched bars).FIG. 8B shows neutralizing antibody response in sera of pigletsvaccinated with no antigen and with 600 micrograms of aluminum phosphate(solid bars) or piglets vaccinated with 50 micrograms of antigen andwith 600 micrograms of aluminum phosphate (hatched bars). Pigletsvaccinated with antigen develop significant levels of serum IgG. Oralchallenge with rotavirus further enhances the serum IgG levels. Thelevels of neutralizing activity are significantly higher in pigletsvaccinated with 50 micrograms of antigen and with 600 micrograms ofaluminum phosphate than mock immunized animals.

FIG. 9A shows virus shedding in fecal samples of piglets vaccinated with50 micrograms of antigen and with 600 micrograms of aluminum phosphate.FIG. 9B shows virus shedding in fecal samples of piglets vaccinated withno antigen and with 600 micrograms of aluminum phosphate. These figuresillustrate that piglets that are mock vaccinated with aluminum phosphateonly or diluent buffer only all shed rotavirus up to 5 days and at hightiter. By contrast, piglets that are vaccinated with inactivatedrotavirus and aluminum phosphate are protected from shedding.

These results show effectiveness of vaccine formulation according toembodiments of the present invention and confirm the results in firstpiglet experiment. These results clearly demonstrate that IRV formulatedwith alum is highly immunogenic and protective against infection inpiglets and consequently establish proof of concept for inactivatedrotavirus vaccine.

Example 7—Gnotobiotic Piglet Model—III—Immunization with CDC-66

To repeat the experiment above using CDC-66 rotavirus, eleven infantgnotobiotic piglets are selected and randomly assigned to 2 groups asindicated in Table 8.

TABLE 8 Piglet Study Design Number of Piglets CDC-66 Antigen aluminumphosphate Group Name in Group (micrograms) (micrograms) GG 5 0 600 HH 650 600

As shown in Table 7, piglets are immunized with a vaccine formulationincluding no antigen and 600 micrograms of aluminum phosphate or 50micrograms of antigen and 600 micrograms of aluminum phosphate (AlPO₄).Each vaccination is carried out by injecting 0.5 milliliters of thevaccine formulation into muscles of the hind legs of the piglets. After3 doses of the vaccine formulation administered at intervals of 10-12days, piglets are orally challenged with virulent DS-1 rotavirus. Priorto virus challenge, each piglet is inoculated with 3 milliliters ofsodium bicarbonate to neutralize acids in the stomach. Fecal specimensare collected from the challenged piglets daily for 10 days. Bloodsamples are collected throughout the experiment at intervals of 7-14days.

Piglets vaccinated with antigen develop significant levels of serum IgG.Oral challenge with rotavirus further enhances the serum IgG levels. Thelevels of neutralizing activity are significantly higher in pigletsvaccinated with 50 micrograms of CDC-66 antigen and with 600 microgramsof aluminum phosphate than mock immunized animals.

Piglets that are mock vaccinated with aluminum phosphate only or diluentbuffer only all shed rotavirus up to 5 days and at high titer. Bycontrast, piglets that are vaccinated with inactivated rotavirus CDC-66and aluminum phosphate are protected from shedding.

These results show effectiveness of vaccine formulation according toembodiments of the present invention and confirm the results in firstpiglet experiment. These results clearly demonstrate that IRV formulatedwith alum is highly immunogenic and protective against infection inpiglets and consequently establish proof of concept for inactivatedrotavirus vaccine.

Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

The invention claimed is:
 1. An immunogenic composition comprising: alive, attenuated human rotavirus or an inactivated human rotavirus, andan adjuvant that increases immunogenicity of the live, attenuated humanrotavirus or inactivated human rotavirus, wherein said live, attenuatedhuman rotavirus or inactivated human rotavirus comprises a NSP1, NSP2,NSP3, NSP4, NSP5, VP1, VP2, VP3, VP4, VP5, VP6 and VP7 protein, andwherein: a) the NSP1 protein has greater than 95% identity to SEQ ID NO:2 or SEQ ID NO: 3; the NSP2 protein has greater than 95% identity to SEQID NO: 5; the NSP3 protein has greater than 95% identity to SEQ ID NO:8; the NSP4 protein has greater than 95% identity to SEQ ID NO: 11; theNSP5 protein has greater than 95% identity to SEQ ID NO: 14 or SEQ IDNO: 15; the VP1 protein has greater than 95% identity to SEQ ID NO: 17;the VP2 protein has greater than 95% identity to SEQ ID NO: 20; the VP3protein has greater than 95% identity to SEQ ID NO: 23; the VP4 proteinhas greater than 95% identity to SEQ ID NO: 26 or SEQ ID NO: 27; the VP6protein has greater than 95% identity to SEQ ID NO: 29 or SEQ ID NO: 30;and the VP7 protein has greater than 95% identity to SEQ ID NO: 32; orb) the NSP1 protein has greater than 95% identity to SEQ ID NO: 71; theNSP2 protein has greater than 95% identity to SEQ ID NO: 77 or SEQ IDNO: 78; the NSP3 protein has greater than 95% identity to SEQ ID NO: 83;the NSP4 protein has greater than 95% identity to SEQ ID NO: 89; a NSP5protein has greater than 95% identity to SEQ ID NO: 95 or SEQ ID NO: 96;the VP1 protein has greater than 95% identity to SEQ ID NO: 101 or SEQID NO: 102; the VP2 protein has greater than 95% identity to SEQ ID NO:107; the VP3 protein has greater than 95% identity to SEQ ID NO: 113 orSEQ ID NO: 114; the VP4 protein has greater than 95% identity to SEQ IDNO: 119 or SEQ ID NO: 120; the VP6 protein has greater than 95% identityto SEQ ID NO: 125; and the VP7 protein has greater than 95% identity toSEQ ID NO:
 131. 2. The immunogenic composition of claim 1, furthercomprising a pharmaceutically acceptable carrier.
 3. The immunogeniccomposition of claim 1, formulated for oral administration to a subject.4. The immunogenic composition of claim 1, formulated for parenteraladministration to a subject.
 5. A method of inducing an immunologicalresponse to a rotavirus in a subject, comprising administering to thesubject an immunogenic amount of the immunogenic composition of claim 1,thereby inducing the immunological response to the rotavirus.
 6. Animmunogenic composition comprising a live, attenuated human rotavirus oran inactivated human rotavirus, wherein said live, attenuated humanrotavirus or inactivated human rotavirus comprises a NSP1, NSP2, NSP3,NSP4, NSP5, VP1, VP2, VP3, VP4, VP5, VP6 and VP7 protein, and wherein:the NSP1 protein has greater than 95% identity to SEQ ID NO: 3, andwherein amino acid 122 is an arginine; the NSP2 protein has greater than95% identity to SEQ ID NO: 5; the NSP3 protein has greater than 95%identity to SEQ ID NO: 8; the NSP4 protein has greater than 95% identityto SEQ ID NO: 11; the NSP5 protein has greater than 95% identity to SEQID NO: 15, and wherein amino acid 45 is an isoleucine; the VP1 proteinhas greater than 95% identity to SEQ ID NO: 17; the VP2 protein has withgreater than 95% identity to SEQ ID NO: 20; the VP3 protein has greaterthan 95% identity to SEQ ID NO: 23; the VP4 protein has greater than 95%identity to SEQ ID NO: 27, and wherein amino acid 51 is a aspartic acid,amino acid 331 is an phenylalanine, amino acid 364 is an isoleucine,amino acid 365 is a histidine, and amino acid 388 is a leucine; the VP6protein has greater than 95% identity to SEQ ID NO: 30, and whereinamino acid 101 is a valine; and the VP7 protein has greater than 95%identity to SEQ ID NO:
 32. 7. The immunogenic composition of claim 6,comprising a pharmaceutically acceptable carrier.
 8. The immunogeniccomposition of claim 6, comprising an adjuvant.
 9. The immunogeniccomposition of claim 6, formulated for oral administration to a subject.10. The immunogenic composition of claim 6, formulated for parenteraladministration to a subject.
 11. A method of inducing an immunologicalresponse to a rotavirus in a subject, comprising administering to thesubject an immunogenic amount of the immunogenic composition of claim 6,thereby inducing the immunological response to the rotavirus.
 12. Theimmunogenic composition of claim 6, wherein the NSP1 protein comprisesthe amino acid sequence of SEQ ID NO: 3; the NSP2 protein comprises theamino acid sequence of SEQ ID NO: 5; the NSP3 protein comprises theamino acid sequence of SEQ ID NO: 8; the NSP4 protein comprises theamino acid sequence of SEQ ID NO: 11; the NSP5 protein comprises theamino acid sequence of SEQ ID NO: 15; the VP1 protein comprises theamino acid sequence of SEQ ID NO: 17; the VP2 protein comprises theamino acid sequence of SEQ ID NO: 20; the VP3 protein comprises theamino acid sequence of SEQ ID NO: 23; the VP4 protein comprises theamino acid sequence of SEQ ID NO: 27; the VP6 protein comprises theamino acid sequence of SEQ ID NO: 30; and the VP7 protein comprises theamino acid sequence of SEQ ID NO:
 32. 13. The immunogenic composition ofclaim 1, wherein the NSP1 protein comprises the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 3; the NSP2 protein comprises the amino acidsequence of SEQ ID NO: 5; the NSP3 protein comprises the amino acidsequence of SEQ ID NO: 8; the NSP4 protein comprises the amino acidsequence of SEQ ID NO: 11; the NSP5 protein comprises the amino acidsequence of SEQ ID NO: 14 or SEQ ID NO: 15; the VP1 protein comprisesthe amino acid sequence of SEQ ID NO: 17; the VP2 protein comprises theamino acid sequence of SEQ ID NO: 20; the VP3 protein comprises theamino acid sequence of SEQ ID NO: 23; the VP4 protein comprises theamino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27; the VP6 proteincomprises the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30; andthe VP7 protein comprises the amino acid sequence of SEQ ID NO:
 32. 14.The immunogenic composition of claim 1, wherein the NSP1 proteincomprises the amino acid sequence of SEQ ID NO: 2; the NSP2 proteincomprises the amino acid sequence of SEQ ID NO: 5; the NSP3 proteincomprises the amino acid sequence of SEQ ID NO: 8; the NSP4 proteincomprises the amino acid sequence of SEQ ID NO: 11; the NSP5 proteincomprises the amino acid sequence of SEQ ID NO: 14; the VP1 proteincomprises the amino acid sequence of SEQ ID NO: 17; the VP2 proteincomprises the amino acid sequence of SEQ ID NO: 20; the VP3 proteincomprises the amino acid sequence of SEQ ID NO: 23; the VP4 proteincomprises the amino acid sequence of SEQ ID NO: 26; the VP6 proteincomprises the amino acid sequence of SEQ ID NO: 29; and the VP7 proteincomprises the amino acid sequence of SEQ ID NO:
 32. 15. The immunogeniccomposition of claim 1, wherein the NSP1 protein comprises the aminoacid sequence of SEQ ID NO: 71; the NSP2 protein comprises the aminoacid sequence of SEQ ID NO: 77 or SEQ ID NO: 78; the NSP3 proteincomprises the amino acid sequence of SEQ ID NO: 83; the NSP4 proteincomprises the amino acid sequence of SEQ ID NO: 89; the NSP5 proteincomprises the amino acid sequence of SEQ ID-NO: 95 or SEQ ID NO: 96; theVP1 protein comprises the amino acid sequence of SEQ ID NO: 101 or SEQID NO: 102; the VP2 protein comprises the amino acid sequence of SEQ IDNO: 107; the VP3 protein comprises the amino acid sequence of SEQ ID NO:113 or SEQ ID NO: 114; the VP4 protein comprises the amino acid sequenceof SEQ ID NO: 119 or SEQ ID NO: 120; the VP6 protein comprises the aminoacid sequence of SEQ ID NO: 125; and the VP7 protein comprises the aminoacid sequence of SEQ ID NO:
 131. 16. An immunogenic compositioncomprising a live, attenuated human rotavirus or an inactivated humanrotavirus, wherein said live, attenuated human rotavirus or inactivatedhuman rotavirus comprises a NSP1, NSP2, NSP3, NSP4, NSP5, VP1, VP2, VP3,VP4, VP5, VP6 and VP7 protein, and wherein: the NSP1 protein has greaterthan 95% identity to SEQ ID NO: 72; the NSP2 protein has greater than95% identity to SEQ ID NO: 78, and wherein amino acid 142 is a tyrosine;the NSP3 protein has greater than 95% identity to SEQ ID NO: 84; theNSP4 protein has greater than 95% identity to SEQ ID NO: 90; the NSP5protein has greater than 95% identity to SEQ ID NO: 96, and whereinamino acid 60 is a valine; the VP1 protein has greater than 95% identityto SEQ ID NO: 102, and wherein amino acid 141 is a serine; the VP2protein has with greater than 95% identity to SEQ ID NO: 10; the VP3protein has greater than 95% identity to SEQ ID NO: 114, and whereinamino acid 365 is a serine; the VP4 protein has greater than 95%identity to SEQ ID NO: 120, and wherein amino acid 254 is a lysine,amino acid 367 is an alanine, amino acid 385 is an asparagine, and aminoacid 392 is a glycine; the VP6 protein has greater than 95% identity toSEQ ID NO: 126, and wherein amino acid 101 is an alanine; and the VP7protein has greater than 95% identity to SEQ ID NO:
 132. 17. Theimmunogenic composition of claim 16, comprising a pharmaceuticallyacceptable carrier.
 18. The immunogenic composition of claim 16,comprising an adjuvant.
 19. The immunogenic composition of claim 16,formulated for oral administration to a subject.
 20. The immunogeniccomposition of claim 16, formulated for parenteral administration to asubject.
 21. A method of inducing an immunological response to arotavirus in a subject, comprising administering to the subject animmunogenic amount of the immunogenic composition of claim 16, therebyinducing the immunological response to the rotavirus.
 22. Theimmunogenic composition of claim 16, wherein the NSP1 protein comprisesthe amino acid sequence of SEQ ID NO: 72; the NSP2 protein comprises theamino acid sequence of SEQ ID NO: 78; the NSP3 protein comprises theamino acid sequence of SEQ ID NO: 84; the NSP4 protein comprises theamino acid sequence of SEQ ID NO: 90; the NSP5 protein comprises theamino acid sequence of SEQ ID NO: 96; the VP1 protein comprises theamino acid sequence of SEQ ID NO: 102; the VP2 protein comprises theamino acid sequence of SEQ ID NO: 10; the VP3 protein comprises theamino acid sequence of SEQ ID NO: 114; the VP4 protein comprises theamino acid sequence of SEQ ID NO: 120; the VP6 protein comprises theamino acid sequence of SEQ ID NO: 126; and the VP7 protein comprises theamino acid sequence of SEQ ID NO: 132.